Compositions and methods for reducing cat allergens in the environment

ABSTRACT

Formulations, compositions and products for reducing allergenic Fel d 1 from the environment are disclosed. The formulations include at least one protease that interacts with the Fel d 1 and substantially degrades allergenic epitopes on the Fel d 1. Methods of using the formulations for reducing or eliminating allergenic Fel d 1 from the environment are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/918,420 filed Dec. 19, 2013, the disclosure of which isincorporated in its entirety herein by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to reducing or preventing cat allergiesor their symptoms, and specifically to compositions and methods forreducing the major cat allergen, Fel 1, from the environment.

2. Description of the Related Art

Domestic cats produce some of the most potent elicitors of allergicreaction, affecting people worldwide. The severity of symptoms rangesfrom mild rhinitis and conjunctivitis to life-threatening asthmaticresponses. The most prominent and potent allergen in cat dander is Feld 1. Fel d 1 elicits IgE responses in 90-95% of patients with catallergy (van Ree et al., 1999, J. Allergy Clin. Immunol. 104: 1223-1230)and accounts for 60-90% of the total allergenic activity in cat dander(Kleine-Tebbe, et al., 1993 Int. Arch. Allergy Immunol. 100: 256-262).

The three-dimensional structures of only a few allergens have beenreported (see Valenta & Kraft, 2001, Immunol. Rev. 179: 119-127). Thebiological functions of these allergens are diverse or unknown, withoutany particular biological or structural feature that seems to predisposea protein to act as an allergen (see Aalberse, 2000, J. Allergy Clin.Immunol. 106: 228-238). Protein structural features believed to berelevant for allergenicity include solubility, stability, size, andcompactness of protein. These aspects reflect dependency ofallergenicity on transport over mucosal barriers and susceptibility toproteases (Aalberse, 2000, supra). Posttranslational modification mayalso affect allergenicity, such as by introducing new epitopes oraltering solubility, stability, size, susceptibility toward proteases,and/or uptake and processing by antigen-presenting cells (Aalberse,2000, supra). Although glycosylation affects many of these processes, itis not determinative of allergenicity in and of itself. Many allergensare not glycosylated, whereas some are heavily glycosylated (Aalberse,2000, supra). As a result, determination of the basis for allergenicitymay require a detailed structural study of the allergen, and even thatmay not yield definitive results.

Fel d 1 is a 35-kDa tetrameric glycoprotein formed by two heterodimers.Each 18-kDa dimer is composed of two covalently linked chains derivedfrom two independent genes, chain 1, comprising 70 residues, and chain 2(of which there are two isoforms), comprising 90 or 92 residues. Thethree-dimensional structure of Fel d 1 has been determined (Kaiser etal., 2003, J. Biol. Chem. 278: 37730-37735, Kristensen et al., 1997,Biol. Chem. 378: 899-908). The fold of the protein was found to bear astriking resemblance to that of uteroglobin, a steroid-inducible,cytokine-like molecule with anti-inflammatory and immunomodulatoryproperties. The relative localization of three IgE epitopes wasdetermined on the molecular surface of Fel d 1, at residues 15-28 (chain2), 117-130, and 138-151 (chain 1), respectively (Kaiser et al., 2003,supra).

Fel d 1 is produced by sebaceous glands and squamous epithelial cellsand is transferred to the fur by licking and grooming (Bartholome etal., 1985, J. Allergy Clin. Immunol. 76: 503-506; Charpin et al., 1991,J. Allergy Clin. Immunol. 88: 77-82; Dabrowski et al., 1990, J. AllergyClin. Immunol. 86: 462-465). Fel d 1 is also present in the salivary,perianal, and lachrymal glands (Anderson et al., 1985, J. Allergy Clin.Immunol. 76: 563-569; van Milligen et al., 1990, Int. Arch. AllergyAppl. Immunol. 92: 375-378). Thus, the allergen is present within and onthe animal, and is also carried by small airborne particles to surfaceswithin the environment occupied by the cat.

Strategies developed for controlling the reactions to such allergensinclude establishing tolerance to the allergen in an individual, andsimple avoidance. Tolerance strategies entail establishment orreestablishment of a less harmful or more productive responses toexogenous allergens. Tolerance-inducing strategies traditionally haveinvolved allergen immunotherapy, wherein the sensitized individual isintentionally exposed to the allergen in a controlled manner, forexample through a series of injections, or through oral or nasalabsorption. Immunotherapy has been in use for over 100 years, and hasbeen successful, although it may take years to establish an acceptablelevel of tolerance. While potentially effective in the specificindividual receiving the treatment, tolerance strategies are expensive,invasive, time-consuming, and require experts such as doctors,immunologists, and the like, for administration. Treatments fortolerance also involve a certain level of risk associated with adversereactions and negative outcomes.

Avoidance of Fel d 1 is attractive in principle, but difficult toachieve. A study of households with cats indicated that Fel D1 is widelypresent, e.g., in 96.6% of the beds, 96.9% of bedroom floors, 96.1% ofliving room floors, and 97.9% of sofas. (Geany et al., Pediatrics,116(2): August 2005). Clothing from school children from homescontaining cats was tested outside of the home (in schools), and foundto contain Fel D1 antigens.

Thus, this environmental antigen poses a substantial risk, not only tosensitized individuals living in households with cats, but to theallergic human population on the whole. (Gerge & Dreborg, Ped. AllergyImmun., 9(1):25-30, 1998). As a result, while cat ownership is on therise in the United States, allergies to cats have become and remain theprimary reason for relinquishment of cats to animal shelters (Scarlettet al., 1999, J. Appl. Animal Welfare Sci. 2:41-57).

Though complete avoidance of Fel dl may not be practicable, a reductionin the amount of Fel d 1, even a minimal reduction, could havesubstantial impact on the health of sensitized individuals. This couldminimize relinquishment as a result of a person in the home becomingsensitized.

Attempts have been made to reduce cat allergens from the environment.For instance, U.S. Pat. No. 7,704,532 discloses methods purported tomitigate allergic reactions in humans and other susceptible animals bydirectly contacting the allergens, including cat allergens, with acomposition containing an acidic salt solution, including salts ofaluminum, calcium and/or magnesium, to clothing, surfaces, interiors,furniture, plant bedding, plants and the like. U.S. Pat. No. 5,826,546discloses a waterless method for shampooing a pet comprising using afoamable shampoo composition in combination with a dispenser capable ofdispensing the composition as a foam. The composition may contain one ormore of: anionic detergents, nonionic detergents, amphoteric detergents,preservatives, antimicrobial agents, antioxidants, mild soaps,surfactants, skin conditioners such as aloe extracts, fragrances, agentsfor treating flea infestation such as melaleuca oil, a pH adjuster suchas citric acid, depending on the particular need of the pet. U.S.2011/0135750 discloses methods and compositions purported to denatureallergens, such as cat Fel d 1, the composition comprising a combinationof calcium salts and lanthanum salts. U.S. 2004/0007251 discloses wetand dry wipe cleaners comprising an additive, such as a lectin, aprotease and/or an enzyme inhibitor purportedly capable of binding to orcleaving an allergen, such as cat Fel d 1, and removing it from asurface. U.S. 2006/0142394 discloses methods for inhibiting dust mitefeces and denaturing animal hair keratin and/or plant pollen or sporesusing composition comprising an enzyme capable of breaking downpolypeptides such that they cannot elicit an allergenic effect onhumans. U.S. Pat. No. 8,454,953 discloses methods for reducing orpreventing allergies or symptoms of allergic reactions to allergenscomprising contacting the source of the allergen with a compositioncomprising a molecule capable of inhibiting the ability of the allergento bind to mast cells in an animal predisposed to having an allergicresponse. The molecule can be an antibody specific for the allergen,such as an antibody specific for the cat Fel d 1 allergen.

Various commercially available products and methods also purport toreduce allergens from the environment. One such method, said to beuseful for prevention or mitigation of allergic reactions in humans,including those caused by cat Fel d 1, is performed by first cleaningsurfaces in the environment and then applying a water-based solutionthat contains an ingredient derived from fruit and vegetable seedextracts, using a spray applicator to surfaces such as mattresses,carpeting, upholstered furniture, rugs and window treatments (seeMASTERBLEND® Special Report on RESPONSIBLECARE SYSTEM™ ALLERGY RELIEFTREATMENT™, url masterblend.net). APDC, Inc. (url apdc-inc.com) producesthe AUER-RX® Anti-Allergen Spray, which is a liquid composition said tobe derived from plants and naturally occurring organic compounds,containing chlorine dioxide. Another method involves directly applying acomposition comprising shampoo and skin conditioning ingredientsdirectly to an animal to remove allergens, including cat Fel d 1, on theanimal's fur and skin (see ALLERPET®, produced by Allerpet, Inc., NewYork, N.Y.). Direct application of the composition to a cat was shown toreduce the amount of Fel d 1 on the cat's fur and on the carpet that wasexposed to the cats (LGH Koren, E Janssen, A Willemse, American Academyof Allergy and Immunology March 1995 Annual Meeting, Eindhoven &Utrecht, Netherlands).

Though, as summarized above, certain methods and compositions areavailable in the art, there remains a need for additional and improvedmethods for reducing Fel d 1 in the environment or rendering it lessallergenic or non-allergenic. There is also a need for compositions thatallow sufficient control of the level and/or potency of Fel d 1 toreduce, minimize, or prevent an allergic response in individualspredisposed to having such a response.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to reduce oreliminate allergenic Fel d 1 from an environment.

It is a further object of the invention to reduce or eliminateallergenic Fel d 1 from environments comprising the local environment ofan individual who may suffer from cat allergies.

It is yet a further object of the invention to reduce or eliminateallergenic Fel d 1 from sources of the allergen, e.g., cat hair, skin orfur, or cat saliva.

One or more of these other objects can be achieved using formulations,products, kits and methods comprising protease enzymes that interactwith Fel d 1 and substantially degrade allergenic epitopes on the Fel d1, thereby reducing or eliminating its allergenicity.

Other and further objects, features, and advantages of the presentinvention will be readily apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. SDS-PAGE showing that Fel d 1 is completely degraded by aBacillus licheniformis subtilisin. Natural Fel d 1 (1.9 μg) wasincubated overnight with 0.1, 1 or 10 rfu s⁻¹ of Bacillus licheniformissubtilisin (Alcalase®, Novozymes) activity at 37° C. either in Tris/HClbuffer (200 mM, pH 7.8) or ammonium acetate buffer (100 mM, pH 4.0. Thesubtilisin (10 rfu s⁻¹) without Fel d 1 and Fel d 1 without thesubtilisin served as controls.

FIG. 2. Histogram showing residual Fel d 1 concentration afterprotease-catalyzed degradation as determined by ELISA. Natural Fel d 1(2.5 μg) was incubated overnight with all tested commercial proteases(except for BLAP) in 20 μl of the optimal buffer at minimally requiredactivities to achieve complete degradation of Fel d 1 (c.f. Table 2).The samples were diluted and analyzed by ELISA with time intervals of 20and 30 minutes. As a result of the dilution, an initial concentration of12 ng ml⁻¹ of Fel d 1 was expected (i.e. in the absence of degradation).The negative control (Neg Ctrl) contained no Fel d 1, the positivecontrols contained Fel d 1 incubated with the different buffers: PosCtrl 1: Tris/HCl buffer (200 mM, pH 7.8), Pos Ctrl 2: sodium phosphatebuffer (100 mM, pH 7.8), Pos Ctrl 3: sodium phosphate buffer (100 mM pH6.0), Pos Ctrl 4: ammonium acetate buffer (100 mM, pH 4.0).Protease-catalyzed degradations were performed in duplicates.

FIG. 3. Histogram showing keratin-hydrolyzing activity of the proteasesas determined by TNBS assay. Keratin powder was incubated overnight withthe hit proteases at concentrations and under reaction conditions thathad been found to be optimal for Fel d 1 degradation (c.f. Table 2).Keratin-degradation products were determined spectrophoto-metrically at405 nm in 10-fold dilutions of the samples after coupling reaction withpicrylsulfonic acid (TNBS). The data represent the difference betweenabsorbance determined in the samples and in controls with the samebuffer but without protease. Average and standard deviation weredetermined from three independent repetitions of the experiment.

FIG. 4. Histogram showing celltoxic effect of proteases on humankeratinocytes as determined by Sulforhodamin B and WST-1 assays.Confluently grown primary human keratinocytes were incubated overnightwith hit proteases (in cell cultivation medium) at concentrations thatwere identified to be minimally required to degrade 125 μg m⁻¹ of Fel d1 (c.f. Table 2). After incubation, remaining cell vitality wasdetermined for all cells per well (adherent and detached cells) bystaining (of metabolically active cells) with WST-1. The remaining cellvitality of the adherent cells alone was then determined by staining (ofthe proteins of living cells) with Sulfrohodamin B. Cells that were notincubated with proteases served as control (corresponds to 100%remaining cell vitality). Average and standard deviation were determinedfrom three independent repetitions of the experiment.

FIG. 5. Histogram showing effect of isopropanol on theprotease-catalyzed degradation of Fel d 1. Hit proteases were incubatedovernight with 0, 2.5%, 5.0%, 7.5%, 10.0% and 12.5% of isopropanol atconcentrations and under reaction conditions that were found to beoptimal for Fel d 1 degradation (c.f. Table 2). The samples were dilutedand analyzed by ELISA with a time interval of 20 minutes. The indicatedresidual Fel d 1 concentration represents the percentage of Fel d 1determined for the samples as compared to the Fel d 1 for the respectivebuffer controls with maximal isorpropanol concentration but withoutprotease. Average and standard deviation were determined from threeindependent repetitions of the experiment.

FIG. 6. Graph showing effect of Tween® 20 on the protease-catalyzeddegradation of Fel d 1. Hit proteases were incubated overnight with 0,10%, 20%, and 30% of Tween® 20 at concentrations and under reactionconditions that were found to be optimal for Fel d 1 degradation (c.f.Table 2). The samples were diluted and analyzed by ELISA with a timeinterval of 20 minutes. The indicated residual Fel d 1 concentrationrepresents the percentage of Fel d 1 determined for the samples ascompared to the Fel d 1 for the buffer controls with the respectiveTween® 20 concentrations but without protease. Average and standarddeviation were determined from three independent repetitions of theexperiment.

FIG. 7. Histogram showing effect of cysteine and differentconcentrations of skim milk on the papain-catalyzed degradation ofdifferent concentrations of Fel d 1 in artificial cat saliva duringovernight incubation at 37° C. 1.25, 12.5 and 125 μg ml⁻¹ natural Fel d1 was incubated overnight with 4.65 μg ml⁻¹ of papain (corresponds to 60rfu s⁻¹ in 20 μl reaction mix, c.f. Table 2) at 37° C. in artificial catsaliva with 0, 71, 714 and 7140 μg ml⁻¹ skim milk with and without 40 mMof cysteine. After incubation, the samples were diluted and analyzed byELISA with a time interval of 30 minutes. As a result of the dilution, amaximal concentration of 12 ng ml⁻¹ of Fel d 1 was expected (i.e., inthe absence of degradation). Average and standard deviation weredetermined from two independent repetitions of the experiment.

FIG. 8. Histogram showing determination of Fel d 1 by ELISA afterincubation for 1 hour and overnight at 37° C. with differentconcentrations of Bacillus licheniformis subtilisin (Protex® 6L) inartificial cat saliva (extreme conditions). 125 μg ml⁻¹ natural Fel d 1was incubated for 0 min, 1 hour or 20 hours (overnight) with 0, 9.5, 95,950 and 9500 μg ml⁻¹ of the subtilisin at 37° C. in artificial catsaliva (extreme conditions). Reaction was stopped by addition of 1 mMPMSF. Samples were diluted and analyzed by ELISA with a time interval of30 minutes. As a result of the dilution, a maximal concentration of 12ng ml⁻¹ of Fel d 1 was expected (i.e., in the absence of degradation).Average and standard deviation were determined from two independentrepetitions of the experiment.

FIG. 9. Histogram showing results of testing papain and Bacilluslicheniformis subtilisin (Protex 6L) at 1000-fold increasedconcentrations for the degradation of Fel d 1 in artificial cat salivaafter incubation for 5 to 60 min. 4.5 mg ml⁻¹ of papain and 9.5 mg ml⁻¹of the subtilisin were incubated for 0, 5, 10, 15 and 60 min with Fel d1 in artificial cat saliva at 37° C. under normal and extremeconditions. Reaction was stopped by addition of 100 μM E64 (for papain)and 1 mM PMSF (for subtilisin). Samples were diluted and analyzed byELISA with a time interval of 30 minutes. The indicated residual Fel d 1concentration represents the percentage of Fel d 1 determined for thesamples as compared to the Fel d 1 for the respective buffer controlswithout protease. Average and standard deviation were determined fromthree independent repetitions of the experiment. Abbr.: n.d., notdetermined

FIG. 10. Histogram showing results of testing different combinations ofpapain and Bacillus licheniformis subtilisin (Protex® 6L) concentrations(at constant ratio) for the degradation of Fel d 1 in artificial catsaliva after incubation for 0 to 60 min. The different combinations ofpapain and subtilisin concentrations (4.5 and 9.5 μg ml⁻¹; 45 and 95 μgml⁻¹; 450 and 950 μg ml⁻¹; 4500 and 9500 μg ml⁻¹ of papain andsubtilisin, respectively) were incubated for 0, 5, 10, 15 and 60 minwith Fel d 1 in artificial cat saliva at 37° C. under normal and extremeconditions. Reaction was stopped by addition of 100 μM E64 and 1 mMPMSF. Samples were diluted and analyzed by ELISA with a time interval of30 minutes. The indicated residual Fel d 1 concentration represents thepercentage of Fel d 1 determined for the samples as compared to the Feld 1 for the respective buffer controls without protease. Average andstandard deviation were determined from two independent repetitions ofthe experiment.

FIG. 11. Histogram showing results of testing different combinations ofpapain and Bacillus licheniformis subtilisin (Protex® 6L) concentrations(with constant papain concentration) for the degradation of Fel d 1 inartificial cat saliva after incubation for 0 to 10 min. The differentcombinations of papain and subtilisin concentrations (4500 and 1188 μgml⁻¹; 4500 and 2375 μg ml-1; 4500 and 4750 μg ml-1; 4500 and 9500 μgml-1 of papain and subtilisin, respectively) were incubated for 0, 5 and10 min with Fel d 1 in artificial cat saliva at 37° C. under normal andextreme conditions. Reaction was stopped by addition of 100 μM E64 and 1mM PMSF. Samples were diluted and analyzed by ELISA with a time intervalof 30 minutes. The indicated residual Fel d 1 concentration representsthe percentage of Fel d 1 determined for the samples as compared to theFel d 1 for the respective buffer controls without protease. Average andstandard deviation were determined from two independent repetitions ofthe experiment.

FIG. 12. Histogram showing results of testing different combinations ofpapain and Bacillus licheniformis subtilisin (Protex® 6L) concentrations(with constant subtilisin concentration) for the degradation of Fel d 1in artificial cat saliva after incubation for 0 to 10 min. The differentcombinations of papain and subtilisin concentrations (565 and 1188 μgml⁻¹; 1125 and 1188 μg ml⁻¹; 2250 and 1188 μg ml⁻¹; 4500 and 1188 μgml⁻¹ of papain and subtilisin, respectively) were incubated for 0, 5 and10 mM with Fel d 1 in artificial cat saliva at 37° C. under normal andextreme conditions. Reaction was stopped by addition of 100 μM E64 and 1mM PMSF. Samples were diluted and analyzed by ELISA with a time intervalof 30 minutes. The indicated residual Fel d 1 concentration representsthe percentage of Fel d 1 determined for the samples as compared to theFel d 1 for the respective buffer controls without protease. Average andstandard deviation were determined from two independent repetitions ofthe experiment.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “allergy” is synonymous with “allergicresponse” or “allergic reaction”. Each of the terms refers to a state ofimmune responsiveness in an animal specific to an exogenous antigen (or“allergen”) that is not otherwise harmful to the animal. A “symptom” ofan allergic response refers to any measure of the aforesaid immuneresponsiveness, e.g. on the molecular level (including measurement of aactivity or expression of a protein, or transcript or gene), thecellular level, organ level, systemic level, or organism level. Suchsymptoms can comprise one or more such levels. Symptoms may includegeneralized phenomena such as inflammation, respiratory complaints,swelling, or distress typically associated with allergy, rhinitis,edema, and allergic skin disorders including but not limited to atopicdermatitis (e.g. eczema), urticaria (e.g. hives) and angioedema, andallergic contact dermatitis. More specific phenomena that are “symptoms”of an allergic response include any measurable or observable change, forexample at the cellular level, including but not limited to local orsystemic changes in cell populations, eosinophilia, recruitment and/oractivation of immune cells, including, for example, mast cells and/orbasophils, changes in antigen-presenting cells (including but notlimited to FccRI-bearing dendritic cells), intracellular or molecularchanges, including measurement or observations of one or more steps inan immunological cascade, release of intracellular compounds thatmediate an allergic response (e.g. mediators), and changes in one ormore cytokines (e.g. IL-3, IL-5, IL-9, IL-4, or IL-13) or relatedcompounds or antagonists thereof. The skilled artisan will understandthat certain symptoms as defined herein as more readily measured thanothers, and some are measured through subjective assessment orself-assessment of the symptom. For other symptoms, there are convenientor rapid assays or measurements for objectively assessing changes.

The term “environment” as used herein has three components as theyrelate to the cat allergen Fel d 1. These are sometimes referred to as“around the cat,” “on the cat” and “in the cat.” The environment “aroundthe cat” refers to a local environment of an individual who may sufferfrom cat allergies, and/or a local environment inhabited by a cat. Forexample a house, room, car, office, hotel, yard, garage, and the like,could each be “environments” as used herein. Any inanimate surface onwhich an allergen may be disposed is considered part of the environment.Airborne particles containing the allergen are also considered part ofthe environment. Although environments around the cat are frequentlyindoors, nothing herein precludes a limited area partially or completelyopen or outdoors to be an environment, for example a patio, deck,landing, lanai, gazebo, porch, or the like can constitute an environmentfor purposes herein. The environment can also comprise a part or all ofan animal that is the source of an allergen, e.g., the cat's skin, furor saliva on the cat's skin or fur (“on the cat”) or the cat's oralcavity or saliva therein (“in the cat”).

As used herein, an “individual” means an individual animal of anyspecies or kind, including a human.

With respect to kits, the term “single package” means that thecomponents of a kit are physically associated in or with one or morecontainers and considered a unit for manufacture, distribution, sale, oruse. Containers include, but are not limited to, bags, boxes or cartons,bottles, packages of any type, design, or material, over-wrap,shrink-wrap, affixed components (e.g. stapled, adhered, or the like), orcombinations of any of the foregoing. For example, a single package kitmay provide containers of individual compositions and/or foodcompositions physically associated such that they are considered a unitfor manufacture, distribution, sale, or use

The term “virtual package” means that the components of a kit areassociated by directions on one or more physical or virtual kitcomponents instructing the user how to obtain the other components,e.g., in a bag or other container containing one component anddirections instructing the user to go to a website or personal deviceapplication (“app”), contact a recorded message or a fax-back service,view a visual message, or contact a caregiver or instructor to obtain,for example, instructions on how to use the kit, or safety or technicalinformation about one or more components of a kit. Examples ofinformation that can be provided as part of a virtual kit includeinstructions for use; safety information such as material safety datasheets; poison control information; information on potential adversereactions; clinical study results; dietary information such as foodcomposition or caloric composition; general information on physicalactivity, exercise, metabolism, endurance and the like.

All percentages expressed herein are by weight of the composition on adry matter basis unless specifically stated otherwise. The skilledartisan will appreciate that the term “dry matter basis” means that aningredient's concentration or percentage in a composition is measured ordetermined after any free moisture in the composition has been removed.

Ranges are used herein in shorthand, so as to avoid having to set out atlength and describe each and every value within the range. Anyappropriate value within the range can be selected, where appropriate,as the upper value, lower value, or the terminus of the range.

Where used herein, the term “about” indicates that the given value, plusor minus 20% or 15% or 10% or 5% or 1%, is intended. “About” is thusused a shorthand to reflect the recognition that small variations fromthe literal value stated are still within the scope of the invention.

As used herein and in the appended claims, the singular form of a wordincludes the plural, and vice versa, unless the context clearly dictatesotherwise. Thus, the references “a”, “an”, and “the” are generallyinclusive of the plurals of the respective terms. For example, referenceto “a cat”, “a method”, or “a product” includes a plurality of such“cats”, “methods”, or “products”. Similarly, the words “comprise”,“comprises”, and “comprising” are to be interpreted inclusively ratherthan exclusively. Likewise the terms “include”, “including” and “or”should all be construed to be inclusive, unless such a construction isclearly prohibited from the context. Where used herein “examples,” or“for example,” particularly when followed by a listing of terms, ismerely exemplary and illustrative, and should not be deemed to beexclusive or comprehensive.

The term “comprising” is intended to include embodiments encompassed bythe terms “consisting essentially of” and “consisting of”. Similarly,the term “consisting essentially of is intended to include embodimentsencompassed by the term “consisting of”.

The methods and compositions and other advances disclosed here are notlimited to particular methodology, protocols, and reagents describedherein because, as the skilled artisan will appreciate, they may vary.Further, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to and does not limitthe scope of that which is disclosed or claimed.

Unless defined otherwise, all technical and scientific terms, terms ofart, and acronyms used herein have the meanings commonly understood byone of ordinary skill in the art in the field(s) of the invention, or inthe field(s) where the term is used. Although any compositions, methods,articles of manufacture, or other means or materials similar orequivalent to those described herein can be used in the practice of thepresent invention, the preferred compositions, methods, articles ofmanufacture, or other means or materials are described herein.

All patents, patent applications, publications, and other referencescited or referred to herein are incorporated herein by reference to theextent allowed by the controlling law. The discussion of thosereferences is intended merely to summarize the assertions made therein.No admission is made that any such patents, patent applications,publications or references, or any portion thereof, is relevant,material, or prior art. The right to challenge the accuracy andpertinence of any assertion of such patents, patent applications,publications, and other references as relevant, material, or prior artis specifically reserved.

Embodiments of the Invention

Compositions, methods, articles of manufacture, kits and packages areprovided for reducing or eliminating the cat allergen Fel d 1 from theenvironment. The invention springs in part from the inventors' discoverythat certain enzymes, particularly proteolytic enzymes (proteases), areable to degrade the antigenic epitopes from Fel d 1, thereby reducing oreliminating its allergenic effect.

One aspect of the invention features a formulation for reducing oreliminating allergenicity of Fel d 1, comprising at least one proteasethat interacts with the Fel d 1 and substantially degrades allergenicepitopes on the Fel d 1. The effective proteases are selected fromserine proteases, thiol proteases, aspartyl proteases, zincmetalloproteases, or any combination thereof.

In certain embodiments, the serine proteases include subtilisin,proteinase K, trypsin, alpha-chymotrypsin, endoproteinase Glu-C andendoproteinase Lys-C. If subtilisin is used, it may be sourced from aspecies of Bacillus selected from B. licheniformis, B. clausii, B.haloudurans, B. lentus, B. amyloliquefaciens, B. subtilis or anycombination thereof. The thiol proteases can include bromelain, papainand ficin, which are obtained from plant sources, e.g., pineapple,papaya and fig plants. The aspartyl proteases can include bovinechymosin, endothiapepsin (e.g., from Chryphonectria parasitica),mucorpepsin/rennin (e.g., from Mucor miehei) pepsin (e.g., of porcineorigin) and aspergillopepsin (e.g., from Trichoderma reesei, Aspergillusniger or Aspergillus oryzae). The zinc metalloproteases can includethermolysin (e.g., from Bacillus thermoproteolyticus rokko orGeobacillus sp.) and endoproteinase Asp-N, (e.g., from Flavobacteriummeningosepticum).

Table 1 below sets forth proteases suitable for use in the presentinvention. The proteases may be purified from a biological source orthey can be obtained from commercial sources. Certain exemplarycommercial sources are shown in the table; many others are available.

TABLE 1 Exemplary Enzyme Functional Protease Origin, RecommendedClassification # Class Tradename Natural Source name (IUPAC) (IUPAC)EINECS # CAS # Serine Alcalase ® 2.4L Bacillus licheniformis Subtilisin3.4.21.62 232-752-2 9014-01-1 Protease (NZ) Savinase ® Bacillus clausiiSubtilisin 3.4.21.62 231-752-2 9014-01-1 16.0L; Everlase ® 16.0L (NZ)Esperase ® 8.0L Bacillus halodurans Subtilisin 3.4.21.62 231-752-29014-01-1 (NZ) Purafect ® (G) Bacillus lentus Subtilisin 3.4.21.62232-752-2 9014-01-1 Protex ® 6L; Bacillus licheniformis Subtilisin3.4.21.62 231-752-2 9014-01-1 Protex ® 8L (G) Protex ® 7L (G) BacillusSubtilisin 3.4.21.62 232-752-2 9014-01-1 amyloliquefaciens Protex ® 30L;Bacillus subtilis Subtilisin 3.4.21.62 232-752-2 9014-01-1 Protex 89L(G) Protex ® 40L (G) Bacillus subtilis Subtilisin 3.4.21.62 231-752-29014-01-1 Tritirachium album Proteinase K 3.4.21.62 Porcine originTrypsin 3.4.4.4 232-650-8 9002-07-7 Bovine origin Alpha- 3.4.21.1232-671-2 9004-07-3 Chymotrypsin Staphylococcus aureus Endoproteinase3.4.21.19 Glu-C Lysobacter Endoproteinase 3.4.21.50 enzymogenes Lys-CThiol Pineapple Bromelain 3.4.22.33 232-572-4 9001-00-7 Protease Caricapapaya Papain 3.4.4.10 232-627-2 9001-73-4 (Papaya) Latex from fig treeFicin 3.4.4.12 232-599-1 9001-33-6 Aspartyl CHY-MAX ® bovine originChymosin 3.4.23.4 Protease (CH) CHY-MAX ® M bovine origin Chymosin3.4.23.4 (CH) Thermolase ® Cryphonectria Endothiapepsin 3.4.23.22 (CH)parasitica Mucor miehei Mucorpepsin/ 3.4.23.23 Rennin Porcine originPepsin 3.4.23.1 232-629-3 9001-75-6 Protex ® 15L (G) Trichoderma reeseiAspergillopepsin 3.4.23.18 232-796-2 9025-49-4 Protex ® 26L (G)Aspergillus niger Aspergillopepsin 3.4.23.18 232-796-2 9025-49-4Protex ® 50FP; Aspergillus oryzae var. Aspergillopepsin 3.4.23.18232-796-2 9025-49-4 Protex ® 51FP (G) Zinc Bacillus thermo- Thermolysin3.4.24.27 232-973-4 9073-78-2 Metallo- proteolyticus rokko proteaseProtex ® 14L(G) Geobacillus sp. Thermolysin 3.4.24.27 232-973-49073-78-3 Flavobacterium Endoproteinase 3.4.24.33 menigosepticum Asp-N(G) = Dupont Industrial Bioscience (formerly Genencor), (NZ) =Novozymes, (CH) = Christian Hansen A/S

In certain embodiments, the protease, or combination of proteases, inthe formulation reduces Fel d 1 binding to anti-Fel d 1 antibodies by atleast 50% under conditions applicable to the protease or combinationthereof, as described in the Examples, as measured by one or more ofELISA, SDS-PAGE, or any other known method for detecting the presence ofthe antigen and/or antigenic epitopes or the binding of the antigen toFel d 1-specific antibodies. For instance, the proteases can include oneor more of subtilisin, proteinase K, alpha-chymotrypsin, trypsin,endoproteinase Lys-C, bromelain, papain, ficin, chymosin,endothiapepsin, mucorpepsin/rennin, pepsin, aspergillopepsin,thermolysin and endoproteinase Asp-N.

More particularly, the protease, or combination of proteases, in theformulation reduces Fel d 1 binding to anti-Fel d 1 antibodies by atleast 90% under conditions applicable to the protease or combinationthereof, as described in the Examples, as measured by one or more ofELISA, SDS-PAGE, or any other known method for detecting the presence ofthe antigen and/or antigenic epitopes or the binding of the antigen toFel d 1-specific antibodies. For instance, the proteases can include oneor more of subtilisin (e.g., from Bacillus licheniformis, B. clausii, B.lentus B. amyloliquefaciens and/or B. subtilis), trypsin (e.g.,porcine), alpha-chymotrypsin (e.g., bovine), bromelain, papain, ficin,chymosin (e.g., bovine), endothiapepsin, mucorpepsin/rennin, pepsin,aspergillopepsin (e.g., from Trichoderma reesei, Aspergillus niger orAspergillus oryzae), thermolysin and endoproteinase Asp-N.

In certain embodiments, the proteases include one or more of papain,subtilisin from B. licheniformis, aspergillopepsin from A. oryzae,endoproteinase Asp-N, bromelain, ficin, alpha-chymotrypsin,endothiapepsin from Cryphonectria parasitica, pepsin and thermolysinfrom Geobacillus sp. In particular embodiments, the proteases includeone or more of papain, subtilisin from B. licheniformis,aspergillopepsin from A. oryzae and endoproteinase Asp-N.

The inventors have determined that certain proteases may be particularlysuitable for use in compositions containing alcohol or certaindetergents. For instance, in certain embodiments, the protease, orcombination of proteases, reduces Fel d 1 binding to anti-Fel d 1antibodies by at least 50% in the presence of up to 7.5% isopropanolunder reaction conditions as set forth for the respective classes ofenzymes in Table 2. These proteases can include one or more of ficin,bromelain, papain, aspergillopepsin from Aspergillus oryzae, orendoproteinase Asp-N. In other embodiments, the protease, or combinationof proteases, reduces Fel d 1 binding to anti-Fel d 1 antibodies by atleast 50% in the presence of non-ionic detergent, e.g., polysorbate 20(Tween® 20) at up to 10%, under reaction conditions as set forth inTable 2. These proteases can include one or more of aspergillopepsinfrom A. oryzae, endoproteinase Asp-N, subtilisin from B. licheniformis,papain, bromelain and ficin. I was thinking the range should be broader?

The formulation can further comprise an additive that enhances theefficiency of the enzyme in degrading the Fel d 1. In certainembodiments, the additive is cysteine or calcium salts/ions (Ca²⁺), or acombination of cysteine and calcium salts/ions, or compounds that formcysteine or calcium salts/ions in situ.

In various embodiments, the formulation is disposed within a compositionselected from, for example: liquid, solid or powder cleaning agent,spray, moist cloth, wipe, sponge, water-dissolvable tablet, filter,food, oil or water supplement, vacuum cleaner filter or additive,granule, detergent, carpet and room deodorizer, litter, litter additive,mitt, additive for non-woven products, washing machine pod (tablet),multi chamber liquid tablets. Additionally, the formulations can bedisposed of in an oral preparation. In one aspect, the formulation canbe disposed within a toy, e.g. cat toy, including edible and non-edibletoys.

In certain embodiments, the formulation can comprise a granule, powderor tablet that is reconstituted with a liquid (e.g., water, buffer orother liquid) prior to use. In other embodiments, the formulation cancomprise a liquid or spray that can be applied to a surface or on theanimal. Preferably, the spray does not aerosolize. Suitable sprays thatdo not aerosolize can be made in accordance with methods known in theart.

In certain embodiments, the formulations contain enzymes that aregenerally recognized as safe for use in foods and cosmetics. Suchformulations are particularly suitable for use in soaps, shampoos,foams/mousses, powders, sprays, conditioners, rinses, gels, lotions,collars, dispersants, moist mitts, wipes, dentifrices and/ormouthwashes, or any other compositions suitable for applying to theskin, hair, fur or oral cavity, or disposed within an ediblecomposition, or formulated for adding to any such compositions.

In certain embodiments, the protease has substantially no interactionwith substances that include keratin, collagen, elastin, fibronectin,other proteins and fibers or fabric (in clothing, carpet, upholstery,curtains and bedding). Suitable proteases that are inactive on suchproteins include, but are not limited to papain and pepsin.

In other embodiments, the protease may be active against keratin and maybe useful in reducing keratin buildup that can occur when the pet isshampooed. For example, bromelain, ficin, aspergillopepsin,endothiapepsin, certain subtilisins, thermolysin, endoproteinase Asp-Nand alpha-chymotrypsin were found to degrade keratin (Example 2).

Another aspect of the invention features a method of making aformulation for reducing or eliminating allergenicity of Fel d 1. Ingeneral, the method comprises combining at least one protease thatinteracts with the Fel d 1 and substantially degrades allergenicepitopes on the Fel d 1 with a medium in which the protease is active orcan be made active prior to use. The medium can be any medium thatsatisfies the aforementioned requirement, including but not limited toliquids, solids, granules, powders, moist cloths, wipes, mitts, sponges,water-dissolvable tablets, filters, foods, dietary supplements,beverages, concentrates to add to foods and beverages, vacuum cleanerfilters or additives, detergents, carpet, upholstery and roomdeodorizers, litters, litter additives, mitts, washing machine pods(tablet), to name just a few.

The proteases can include any of the proteases discussed hereinabove, orany combination thereof, in an amount suitable to degrade the Fel d 1sufficiently to inhibit it from binding to anti-Fel d 1 antibodiesduring the time the formulation is exposed to the Fel d 1 in theenvironment (around the cat, on the cat or in the cat). For instance,formulations intended to be applied to an environment and removedshortly thereafter (e.g., within minutes or hours), such as a cleaningagent, pet shampoo or oral product, should contain a concentration ofprotease(s) sufficient to degrade Fel d 1 within that time period. Thekinetic analyses discussed in the Examples provide concentration rangesof the proteases to achieve such a result. For example, kinetic analysisof papain and subtilisin from B. licheniformis in artificial cat salivarevealed that, under normal cat saliva conditions, more than 80% of Feld 1 was degraded within 5 minutes by papain at 4.5 mg ml⁻¹ and by thesubtilisin at 9.5 mg ml⁻¹. Formulations intended to remain in place foran extended time (e.g., several hours or overnight), such as anupholstery or fabric spray or a leave-in mousse, gel, shampoo or spray,would require a lesser concentration of protease(s).

The skilled artisan can measure Fel d 1 degradation by any method knownin the art. For example, degradation of Fel d 1 can be measured by ELISAand/or SDS-PAGE, or combinations thereof. In a particular embodiment,ELISA assays are used to determine the amount of binding of Fel d 1 toFel d 1—specific antibodies following exposure to the protease(s).

In one embodiment, protease activity is measured and standardized usinga standard protease assay. Using such an assay, protease activities canbe standardized across proteases and assay conditions. A typicalprotease activity assay system includes a protease substrate and asuitable buffer to support protease activity, and may also includediluents or solvents and other agents useful for the activity ofproteases (e.g., cysteine). Typically, the substrate is designed suchthat cleavage of the substrate by the protease generates a detectableproduct. For instance, one type of protease assay utilizes a protein,e.g., casein, derivatized to contain fluorophore that is quenched untilthe protease cleaves the protein. Upon cleavage by the protease, thefluorophore is separated from the quencher and yields a quantitativelydetectable fluorescence signal.

Other types of protease activity assays are also suitable for use. Forinstance, an assay may utilize a succinylated protein, such as casein,as a substrate. Hydrolysis of this substrate in the presence of proteaseresults in the release of peptide fragments with free terminal aminogroups. These peptides are reacted with trinitrobenzene sulfonic acid(TNBS), followed by measurement of the absorbance increase that resultsfrom the formation of yellow colored TNB-peptide adducts.

The enzyme activities determined by a protease assay can be expressedgenerally as relative product units per second and normalized to theamount of enzyme preparation (rpu s⁻¹ g⁻¹). In the case of afluorescence-based assay such as the one described above, the enzymeactivities can be expressed as relative fluorescence units per secondand normalized to the amount of enzyme preparation (rfu

The method comprises combining the requisite amount of the protease withthe medium. The amount of protease to include in the formulation willdepend on whether it is prepared in “ready to use” form, or as aconcentrate for later dilution.

Another aspect of the invention features a method of reducing oreliminating allergenic Fel d 1 from the environment. The methodcomprises contacting an element of the environment where Fel d 1 ispresent with a formulation comprising at least one protease thatinteracts with the Fel d 1 and substantially degrades allergenicepitopes on the Fel d 1, thereby reducing or eliminating allergenic Feldl from the environment.

In certain embodiments, the environment is “around the cat.” In oneembodiment, the Fel d 1 is present on an inanimate surface and theformulation is applied to the surface. Typical surfaces can includecounters, floors, walls, furniture, upholstery and clothing, to name afew. In another embodiment, the Fel d 1 is airborne and the formulationis contacted with the air. For instance, the formulation can be disposedwithin a filter through which air passes, such as an air filter for afan, heater or air conditioner, or a vacuum cleaner filter.

In other embodiments, the environment is “on the cat.” and theformulation is applied to the portion of the animal on which the Fel d 1is present. For instance the Fel d 1 may be present on the hair, fur orexternal skin of the animal, or on saliva deposited on the hair, fur orskin of the animal. In other embodiments, the environment is “in thecat,” typically in the mouth of the animal where Fel d 1—containingsaliva is produced, and the formulation is applied as a dentifrice,rinse, food, treat, film or strip foam or spray or beverage.

Another aspect of the invention features an article of manufacture (alsoreferred to herein as a “product”) comprising a formulation thatincludes at least one protease that interacts with the Fel d 1 andsubstantially degrades allergenic epitopes on the Fel d 1, andinstructions for its use in reducing or eliminating allergenic Fel dlfrom the environment. In one embodiment, the product can be formulatedas a liquid, solid or powder cleaning agent, spray, moist cloth, wipe,sponge, water-dissolvable tablet, detergent, carpet or fabricdeodorizer, litter, litter additive, mitt, additive for non-woven orwoven products, washing machine pod (tablet), multicompartment liquidtablet, for application to an inanimate surface. In another embodiment,the product can be formulated as an air filter additive for contactingairborne Fel d 1.

In other embodiments, the product is formulated for application oradministration to an animal that produces the Fel d 1. For instance, theproduct can be formulated as a shampoo, conditioner, rinse, mousse, gel,spray, lotion or powder for application to hair, fur or external skin ofthe animal. Alternatively, the product can be formulated as adentifrice, food, treat, or additive to the animal's food or water, forapplication to the mouth of the animal.

Such products and articles of manufacture as disclosed herein can beeffective for inactivating Fel d 1 in an environment. In one embodiment,the products and/or articles can reduce or inactivate Fel d 1 by atleast 10% in their respective environments. In some aspects, Fel d 1 canbe inactivated by the products and/or articles by at least 50%. In otheraspects, Fel d 1 can be inactivated by at least 1%, 5%, 15%, 20%, 30%,40%, 60%, 70%, 80%, 90%, or even by at least 95%.

In certain embodiments, the product comprises a food or other ediblecomposition for reducing or eliminating the allergenicity of the catallergen Fel d 1. In one embodiment, the food product is a dry pet foodor pet treat comprising a protease-containing formulation. For example,the formulation can be applied by dusting or coating the formulationonto the dry food composition prior to packaging or shipping. Becausethe food product is dry, activity of the proteases can be preservedduring shipping and storage. The formulation can also be provided as aconcentrate that is dissolved prior to feeding, or a sachet or pouchcontaining a powdered or granular protease preparation that can besprinkled onto a food composition or mixed into water or other liquidbeverages. The formulation can be provided as a liquid formulation thatcan be applied, for example, directly to a food composition (dry, moistor intermediate), or to water or other liquid beverages.

In a further aspect, the present invention provides kits for reducing oreliminating the allergenicity of the cat allergen Fel d 1. In general,these kits comprise one of the aforementioned protease-containingformulations and instructions for their use in removing allergenic Fel d1 from the environment.

In one embodiment, the kit comprises a composition for cleaning orotherwise removing Fel d 1 from the environment around the cat, such asa surface of an inanimate object. These include, for instance, a liquid,solid or powder cleaning agent, spray, moist cloth, wipe, sponge, carpetand room deodorizer, granule or detergent, said composition comprisingthe Fel d 1-degrading formulation, along with instructions for use. Incertain embodiments, the protease formulations can be provided inconcentrated form and the instructions will contain directions fordilution. In other embodiments, a multi-component kit is provided. Forexample, a cleaning kit can comprise a cleaning agent in one containerand a protease formulation in another, and the instructions can directthe user on how to combine the components prior to use. Such embodimentscan be particularly beneficial if the proteases in the formulation aresensitive to ingredients in the cleaning agent, such that they would beinactivated by the cleaning agent with prolonged exposure.

Likewise, the kit may comprise a composition for washing fabric, such asa liquid, solid or powder, water-dissolvable tablet or washing machinepod (tablet), said composition comprising the Fel d 1-degradingformulation, along with instructions for use. In another embodiment, thekit comprises an air filter, such as a vacuum cleaner filter oradditive, comprising the Fel d 1-degrading formulation, along withinstructions for use. In these embodiments, the protease formulationsagain can be provided in concentrated form and the instructions cancontain directions for dilution. In other embodiments, a multi-componentkit is provided. For example, a laundry kit can comprise a laundrydetergent in one container and a protease formulation in another, andthe instructions can direct the user on how to combine the componentsprior to use. An air filter kit can comprise the air filter and theprotease formulation, and instructions for combining the two to reduceor eliminate airborne Fel d 1.

In another embodiment, the kit comprises treatments for “on the cat,”e.g., fur, hair or skin. Such kits can include a soap, shampoo, powder,spray, conditioner, rinse, mousse, gel, lotion, collar, dispersant ormoist mitt or wipe suitable for applying to the skin, hair or fur orformulated for adding to any such compositions, along with instructionsfor use. The protease-containing formulation can be included within thefur/hair treatment composition, or it can be provided separately, as aconcentrate or otherwise, and mixed with the fur/hair treatment prior touse. The kits can also contain a combination of fur/hair treatments. Forinstance, a kit can comprise a mitt or cloth for wiping the fur and aliquid protease formulation. The instructions can provide directions onhow to impregnate the mitt or cloth with the liquid and apply it to thecat. As another example, a kit can comprise a shampoo and a rinse,spray, gel or mousse wherein the shampoo is a standard pet shampoo thatdoes not contain the protease formulation and the rinse, spray, gel ormousse contains the protease formulation.

In another embodiment, the kit comprises an oral composition, such as aliquid, solid or powder, moist cloth, wipe, dentifrice or mouthwash,said composition comprising the Fel d 1-degrading formulation, suitablefor applying to the oral cavity, or formulated for adding to any suchcompositions, along with instructions for use. In another embodiment,the kit comprises a dental kit comprising a water-dissolvable tabletthat can be administered to the animal, such as by dissolving indrinking water.

In another embodiment, the kit comprises an edible form of a compositiondescribed herein in a sachet or pouch attached to or recommended to beused alongside to a food composition, such as a pet food package, alongwith instructions for mixing the edible composition into the food,adding the composition to the food, or dissolving, mixing, or adding thecomposition to a fluid that is to be administered to the animalreceiving the food, such as drinking water. In another embodiment, thekit comprises at least a food composition described herein comprisingthe Fel d 1-degrading formulation, along with instructions for use. Inanother kit, a concentrated form of the composition is provided, andalso provided is a tool or device for conveniently measuring a suitableamount of the concentrate for mixing with, adding to, diluting, ordissolving with a food or fluid to be provided to the animal. In onekit, the composition in edible form is provided in a convenient dosagein a series of identical packages, such that one package of thecomposition is added to one package (e.g. can) of pet food without arequirement for measuring. Such kits can be provided such that for eachpackage of pet food in a point-of-sale package, there is one package ofFel d 1-degrading formulation provided. For example, twelve cans of foodand twelve packages of composition are packaged together in a singlekit.

In any of the foregoing embodiments, the kit can comprise a composition,in concentrated or other form, instructions for use, including, ifrequired, instructions for preparation of a suitable dilution, andoptionally one or more of a diluent or extender, a tool or measuringdevice for preparing a suitable dilution, and an applicator such as asprayer, duster, wipe, or the like. Such kits may be useful forcompositions formulated for treating surfaces, for treating the air inan environment, or for treating an animal with a composition forexternal use.

For all such kits, the kits may include devices, applicators, dilutors,and the like that are automatic or the automate the dosing, dilution,mixing, addition, or application of the composition for an appropriateuse. For any of the kits described herein, they can be provided assachets or bundled with other products to maximize convenience,compliance, and efficiency of use and purchase. Thus, the kits caninclude, or be bundled with any or all of food for the pet, bedding forpet, shampoo or cosmetic items for the pet, medicine for the pet.

Any of the foregoing kits, as well as others can also be provided asvirtual kits. When the kit comprises a virtual package, the kit providesinstructions in a virtual environment in combination with one or morephysical kit components, such as those described above. The kit containsat least one composition described herein, and other components,including optional components. The kits may contain the kit componentsin any of various combinations and/or mixtures. In one embodiment, thekit contains a packet containing one or more compositions and acontainer of food for consumption by an animal. The kit may containadditional items such as a device for mixing the compositions andingredients or a device for containing the admixture, e.g., a food bowl.In another embodiment, the compositions are mixed with additionalnutritional supplements such as vitamins and minerals that promote goodhealth in an animal. Further information and instructions are providedin the virtual environment that is provided to the purchaser—i.e.directions to a website, faxback server, or an included computerreadable device such as a CD-ROM and/or an application (“app”) for adevice.

In another aspect, the invention provides a communication means, or ameans for communicating information about or instructions for one ormore of the formulations, methods, compositions, articles ofmanufacture, products and/or kits described herein for reducing theallergenicity of, or the amount of allergenic Fel d 1 from theenvironment. In various embodiments, the information pertains toformulations, compositions, articles of manufacture or products of thepresent invention. In other embodiments, the information pertains tomethods or kits useful for practicing the invention described herein. Inother embodiments, the information relates to combinations of any of theforegoing.

The communication means comprises one or more of text information, audioinformation, still or moving images, including animations, or video. Invarious embodiments, the communication means comprises one or more of aprinted document, a static or dynamic electronic document, for example ahypertext document, a computer readable or digital storage medium,including but not limited to electronic, optical, or magnetic media ofany type, audio information, an audio, audiovisual or visual display orpresentation, or video information however encoded, wherein thecommunication means displays or contains information or instructionscomprising any of the aforesaid. In certain embodiments, thecommunication means comprises a web site, an FAQ (Frequently AskedQuestions) page or file, an electronic file or collection of two or moreelectronic files of the same or different types, an email or email file,a visual display, kiosk, brochure, advertisement, package or productlabel, package or product insert, handout, public announcement,audiotape or electronic audio file embodied in any machine-readable orcomputer-readable medium, a videotape, videodisk, or electronic videofile embodied in any machine readable or computer-readable medium, DVD,CD-ROM, app, or the like, or any combination of the foregoing containingsuch information or instructions. Useful information includes one ormore of (1) methods and techniques for combining and administering theallergen-specific molecules and/or other components, (2) contactinformation for allergic animals or their guardians or caregivers to useif they have a question about the kit, the composition, or its use; (3)nutritional information about food compositions, and other componentsprovided in any kit, (4) safety information including for exampleemergency information, and further contacts in the event of adversereaction; poison control, material data safety sheets, (5) informationuseful for reordering, for example through automatic fulfillmentsystems; (6) general information about allergies, environmentalallergens, and methods for minimizing or eliminating specificenvironmental allergens. Useful instructions can include amounts formixing and administration amounts and frequency. The communication meansis useful for instructing on the benefits of using the present inventionand communicating the approved methods for administering the inventionto an animal.

Another aspect of the invention provides a package comprising any one ormore of the formulations, compositions, products and/or kits describedherein. The package has affixed thereto a label containing a word orwords, picture, symbol, design, acronym, slogan, phrase, or otherdevice, or combination thereof (the label “device”), that indicates thatthe contents of the package contains a protease formulation for reducingor eliminating allergenic Fel d 1 from the environment.

EXAMPLES

Various aspects of the invention can be further illustrated by thefollowing examples. It will be understood that these examples areprovided merely for purposes of illustration and do not limit the scopeof the invention disclosed herein unless otherwise specificallyindicated.

Example 1

A set of 32 proteases, in combination with chemical agents such ascysteine, were tested for their ability to degrade Fel d 1. While notintending to be limited to a particular mechanism, the rationale forenzyme-catalyzed inactivation of Fel d 1 was to determine whichproteases could degrade Fel d 1 such that IgE antibodies would not beable to recognize and bind to Fel d 1, resulting in the failure to mountan immune response and allergic reaction. Candidate proteases wereselected from several different families of proteases, including serineproteases, thiol proteases, aspartic acid proteases and zincmetalloproteases.

To standardize activities and identify optimal reaction conditions foreach candidate protease, the ENZCHEK® Protease Assay Kit (greenfluorescence) from Invitrogen, Inc. (Carlsbad Calif.) was used, which isbased on casein as substrate, derivatized to contain fluorophore that isquenched until cleaving by the protease. The enzyme activitiesdetermined by the protease assay were expressed as relative fluorescenceunits per second and normalized to the amount of enzyme preparation (rfus⁻¹ g⁻¹). To identify appropriate reaction conditions for the differentclasses of enzymes, different buffers and pH conditions were tested at37° C. Table 2 provides the pH of the buffer with the best performance,and the activity (increase of fluorescence per second, rfu s⁻¹) peramount of protease preparation (g⁻¹) as determined by the ENZCHEK®Protease Assay.

Most of the proteases tested (serine proteases, thiol proteases andzinc-metalloproteinases) showed optimal activity in reaction buffers atpH 7.8. The aspartic acid proteases were optimally active at pH 4.0 orpH 6.0. Some serine and aspartic acid proteases were activated by theaddition of calcium salt/ions (Ca²). The thiol proteases weresignificantly more active when cysteine was added to the reactionbuffer. Endoproteinase Lys-C was the only protease that was not activein the ENZCHEK® Protease Assay under the tested conditions.

To characterize Fel d 1 degradation, different concentrations ofcandidate proteases were incubated with Fel d 1 at 37° C. overnight (-18hours), followed by separation of proteins by gel electrophoresis(SDS-PAGE under non-reducing conditions) and COOMASSIE® staining. Atypical reaction mix to test Fel d 1 degradation contained 2.5 μg ofnatural Fel d 1 (nFel d 1, LOTOX® Natural Feld d 1 was obtained fromIndoor Biotechnologies (LTN-FD1-1)) and varying concentrations ofcandidate proteases in a total reaction volume of 20 μl (final Fel d 1concentration of 125 μg ml⁻⁴). Fel d 1 has been reported to be a 35 kDatetrameric glycoprotein formed by two heterodimers of 18 kDa. Therefore,Fel d 1 degradation was assessed by the extent of disappearance of theFel d 1 protein band at approximately 18 kDa. For most proteases,conditions were optimized to promote complete degradation of Fel d 1with minimal protease activity. For all tested proteases, the minimalactivity (rfu s⁻¹) as well as the corresponding amount of enzyme (μg)that was required to completely degrade 2.5 μg Fel d 1 in 20 μl ofbuffer (with optimal pH for the respective protease) was estimated basedon the absence of the Fel d 1 band on the SDS-PAGE gel. The results aresummarized in Table 2.

TABLE 2 Characterization and activity of proteases ActivityEnzChek ®^(c) Min. req. activity^(e) Name or trade nameDescription/Source^(a) Manufacturer^(b) [rfu s⁻¹ g⁻¹] Opt. pH^(d) [rfus⁻¹] [μg] Serine proteases Alcalase ® 2.4L Subtilisin B. licheniformis≧2.4 U g⁻¹ 2.39 * 10⁷ pH 7.8 1-10 0.04-0.42 Savinase ® 16.0L SubtilisinBacillus sp. ≧16 U g⁻¹ 5.34 * 10⁸ pH 7.8 ~100 ~0.19 Everlase ® 16.0LSubtilisin Bacillus sp. ≧16 U g⁻¹ 9.92 * 10⁷ pH 7.8 100-1000 1.0-10 Esperase ® 8.0L Subtilisin from Bacillus sp. ≧8 U g⁻¹ 1.08 * 10⁹ pH 7.8~1000 ~0.93 Purafect ® Subtilisin n.a. 2.40 * 10⁸ pH 7.8 >100 >0.42Protex ® 6L Subtilisin B. licheniformis 7670 U g⁻¹ 5.33 * 10⁸ pH 7.810-100 0.02-0.19 Protex ® 7L Subtilisin B. amyloliquefaciens 265 U g⁻¹1.51 * 10⁵ pH 7.8 <1 <6.6 Protex ® 8L Subtilisin B. licheniformis 4760 Ug⁻¹ 4.44 * 10⁸ pH 7.8 ~100 ~0.22 Protex ® 30L Subtilisin B. subtilis1745 U g⁻¹ 2.01 * 10⁷ pH 7.8 10-100 0.50-5.0  Protex ® 40L Subtilisin B.subtilis 3525 U g⁻¹ 4.97 * 10⁸ pH 7.8 ~100 ~0.20 Protex ® 89L SubtilisinB. subtilis 1930 U g⁻¹ 1.53 * 10⁷ pH 7.8 10-100 0.65-6.5  BLAP (Henkel)Subtilisin B. lentus n.a. 2.40 * 10⁸ pH 7.8 >100 >0.42 Proteinase KSubtilisin Tritirachium album 38.4 U g⁻¹ 3.22 * 10⁷ pH 7.8 ~1000 ~31Trypsin porcine pancreas 1-2 MU g⁻¹ 1.95 * 10⁷ pH 7.8 Ca 10-1000.51-5.1  α-Chymotrypsin porcine pancreas ≧40 kU g⁻¹ 2.35 * 10⁸ pH 7.8Ca 10-100 0.04-0.42 Endoproteinase Glu-C V8 protease S. aureus ~750 kUg⁻¹ 5.95 * 10⁶ pH 7.8 not active (>16 μg) Endoproteinase Lys-CLysobacter enzymogenes n.a. not active not active (>0.5 μg) Thiolproteases Bromelain Pineapple 500 U g⁻¹ 7.35 * 10⁸ pH 7.8 Cys 270-27000.37-3.7  Papain papaya >3 * 10⁷ U g⁻¹ 6.40 * 10⁸ pH 7.8 Cys ~60 ~0.093Ficin fig tree latex 200 U g⁻¹ 1.69 * 10⁹ pH 7.8 Cys 460-4600 0.28-2.8 Aspartic acid proteases CHY-MAX ® Chymosin n.a. 1.58 * 10⁴ pH 4.0Ca >10 >630 CHY-MAX ® Chymosin n.a. 3.96 * 10³ pH 4.0 not active (>2.5mg) Thermolase ® Chymosin C. parasitica n.a. 1.59 * 10⁶ pH 4.0 Ca ~10~6.3 Mucorpepsin Chymosin Mucor miehei 120 U g⁻¹ 4.07 * 10⁶ pH4.04.07-41 1.0-10   Pepsin porcine pancreas 800 U g⁻¹ 2.55 * 10⁶ pH4.02.55-26 1.0-10   Protex ® 15L Trichoderma reesei 31 U g⁻¹ 3.40 * 10⁶pH 4.0~10 2.9 Protex ® 26L Aspergillus niger 190 U g⁻¹ 5.94 * 10⁶ pH4.0~10 1.7 Protex ® 50FP Aspergillus oryzae var. 105 U g⁻¹ 2.39 * 10⁶ pH4.0~1 0.42 Protex ® 51FP Aspergillus oryzae var. 1355 U g⁻¹ 2.21 * 10⁷pH 6.0~100 4.5 Zinc-metallopinteases Thermolysin B. thermoproteolyticusrokko 50-100 kU g⁻¹ 2.13 * 10⁸ pH 7.8 not active (>4.7 μg) Protex ® 14L(Gr) Thermolysin Geobacillus sp. 38 U g⁻¹ 2.92 * 10⁶ pH 7.8 500 ~171Endoproteinase Asp-N Flavastacin 25 kU g⁻¹ 2.24 * 10⁷ pH 7.8^(f) 50 2.2^(a)Name of abbr. organisms: Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus lentus, Staphylococcus aureus, Cryphonectriaparasitica, Bacillus thermoproteolyticus rokko ^(b)Activity of theprotease lot as indicated on the datasheet of the manufacturer.Activities (in U g⁻¹) are determined by variable methods and thus cannot be directly compared ^(c)Activity (increase of fluorescence persecond, rfu s⁻¹) per amount of protease preparation (g⁻¹) as determinedby the EnzChek ® protease assay (Invitrogen, E6638) ^(d)pH of thereaction buffer at which highest activities were determined by theEnzChek ® protease assay. Ca, addition of calcium. Cys, addition ofcysteine ^(e)Minimal activity (as determined in the EnzChek ® proteaseassay (in rfu s⁻¹) and the corresponding amount of protease preparation(in μg) that is required to completely degrade 2.5 μg of natural Fel d 1in 20 μl reaction mix as determined by SDS-PAGE (see text for details).^(f)The buffer that was supplemented by the manufacturer was used forthe EnzChek ® protease assay as well as for Fel d 1 degradation

Various serine proteases candidates were tested for their ability todegrade Fel d 1. For example, the Fel d 1 proteolytic activity of asubtilisin from B. licheniformis (bl) (ALCALASE® 2.4L, Novozymes A/SBagsvaerd, Denmark) was determined. Natural Fel d 1 (1.9 μg) wasincubated with 0.1, 1 or 10 rfu s⁻¹ of the bl subtilisin overnight at37° C. in 20 μl of either Tris/HCl buffer (200 mM, pH 7.8) or ammoniumacetate buffer (100 mM, pH 4.0). The subtilisin (10 rfu s⁻¹) without Feld 1 and Fel d 1 without the subtilisin served as controls. Fel d 1 wascompletely degraded by 10 rfu s⁻¹ of the bl subtilisin in Tris/HClbuffer, but not in ammonium acetate buffer conditions as determined bySDS-PAGE and COOMASSIE® staining (FIG. 1). These experiments revealedthat the minimum amount of bl subtilisin required to completely degrade1.9 μg Fel d 1 in 20 μl Tris/HCL buffer is 1-10 rfu s⁻¹ (0.04-0.42 μg).

Subtilisins from B. clausii (bc) (SAVINASE 16.0L, EVERLASE® 16.0L,Novozymes) and B. halodurans (bh) (ESPERASE® 8.0L, Novozymes) were alsoassessed to determine the minimum amount of protease required to degradeFel d 1. In these experiments, natural Fel d 1 (2.5 μg) was incubatedwith 100 or 1000 rfu s⁻¹ of the respective subtilisin overnight at 37°C. in 20 pl sodium phosphate buffer (100 mM, pH 7.8). Protease (1000 rfus⁻¹) without Fel d 1 and Fel d 1 without protease served as controls.The minimum amounts of the respective proteases required for degradationof 2.5 μg Fel d 1 in 20 μl are shown in Table 2.

The vegetable thiol proteases bromelain, papain and ficin from theedible plants pineapple, papaya and fig tree, respectively, were assayedfor degradation of Fel d 1. For the SDS-PAGE experiments, natural Fel d1 (2.5 μg) was incubated overnight at 37° C. with different amounts ofpapain, bromelain or ficin in sodium phosphate buffer (100 mM, pH 7.8)containing 40 mM cysteine. Buffer with cysteine alone, enzyme withoutFel d 1 in cysteine-free buffer and Fel d 1 without enzyme (with andwithout cysteine) served as controls. The thiol proteases bromelain,papain and ficin were all able to completely degrade Fel d 1 asdetermined by SDS-PAGE and COOMASSIE® staining. As shown in Table 2, theoptimal conditions for bromelain, papain and ficin determined by theENZCHEK® Protease Assay include a pH of 7.8 in the presence of cysteine.Specifically, the addition of 40 mM cysteine increased the activities ofeach of bromelain, papain and ficin by factors of 270, 60 and 460,respectively, as determined by the ENZCHEK® Protease Assay. Table 2shows the minimum amounts of these proteases required for degradation of2.5 μg Fel d 1 in 20 μl cysteine-containing buffer.

In addition, the aspartic acid proteases endothiapepsin fromCryphonectria parasitica (THERMOLASE®, Chr. Hansen, Hφrsholm, Denmark)and chymosin of bovine origin (CHY-MAX®, Chr. Hansen, Hφrsholm, Denmark)were both optimally active at pH 4.0 in the presence of calciumsalt/ions (Ca²⁺) (Table 2). Natural Fel d 1 (2.5 μg) was incubatedovernight at 37° C. with 0.1, 1 or 10 rfu s⁻¹ of the proteases in 20 μlof ammonium acetate buffer (100 mM, pH 4.0) containing 10 mM calciumchloride. Buffer without Fel d 1 and Fel d 1 without enzyme served ascontrols in this experiment. Table 2 shows the minimally requiredprotease amounts for degradation of 2.5 μg Fel d 1 in 20 μl buffer forthe two aspartic acid proteases.

Some of the candidate proteases were tested for their abilities todegrade 2.5 μg of natural Fel d 1 in 20 μl optimal reaction buffer at37° C. within short incubation time points by adding a proteaseinhibitor after one or two hours to stop the reaction. Reactions werevisualized by SDS-PAGE followed by COOMASSIE® staining. It was foundthat 50 rfu s⁻¹ (2.1 μg) of a bl subtilisin (ALCALASE® 2.4L) wassufficient to completely degrade Fel d 1 within one hour of incubationwhen using 1 mM phenylmethylsulfonyl fluoride (PMSF) as inhibitor.Moreover, 600 rfu s⁻¹ (0.93 μg) of papain degraded most Fel d 1 afterone hour of incubation when using 10 E64 as inhibitor. 500 rfu s⁻¹ (171μg) of thermolysin from Geobacillus sp. (PROTEX® 14L, DuPont IndustrialBiosciences) was sufficient to completely degrade 2.5 μg Fel d 1following overnight incubation, but only partial degradation occurredafter two hours incubation when using 10 mM ethylenediaminetetraaceticacid (EDTA) as inhibitor. The kinetic studies using protease inhibitorsrevealed that complete Fel d 1 degradation generally could be achievedin a shorter time by increasing the protease concentration. Likewise,greater amounts of Fel d 1 could be degraded by increasing the proteaseconcentration.

To summarize, the results from the Fel d 1 proteolysis experiments usingthe panel of 32 protease candidates revealed that proteases from allclasses were able to completely degrade Fel d 1. As shown in Table 2,the candidate proteases exhibited varying degrees of Fel d 1 degradationactivities. Most candidate proteases were capable of degrading 2.5 μgFel d 1 in 20 μl at an activity less than 100 rfu s⁻¹.

Next, the ability of Fel d 1 specific antibodies to recognize and bindFel d 1 after proteolysis was determined by ELISA. Natural Fel d 1 andFel d 1 specific antibodies were obtained from Indoor BiotechnologiesLtd (Indoor Biotechnologies Ltd (Cardiff, Wales). ELISA conditions weredetermined such that a reliable quantitation of natural Fel d 1 in arange of concentrations from 0.3 to 12 ng ml⁻¹ was possible. Variouscandidate proteases, as indicated in FIG. 2, were incubated with Fel d 1(2.5 μg) overnight in 20 μl of the optimal buffer at minimally requiredactivities to achieve complete degradation of Fel d 1 (See optimizedconditions reported in Table 2). Samples were diluted to a finaltheoretical Fel d 1 concentration of 12 ng ml⁻¹ (initial substrateconcentration) and analyzed by ELISA at two different assay reading timepoints (20 and 30 minutes). After incubating Fel d 1 with a candidateprotease, plates coated with anti-Fel d 1 antibody were used to captureFel d 1. Then, an enzyme-linked detecting antibody that binds Fel d 1was added, followed by the addition of substrate, which wasenzymatically converted to a detectable signal. In the event that theproteases degrade Fel d 1, such that Fel d 1 epitopes required forbinding to anti-Fel d 1 antibody are no longer present and/oraccessible, the anti-Fel d 1 antibodies would not bind Fel d 1 and nosignal would be detected by ELISA. As a control to verify that proteaseactivity was not interfering with the ELISA assay, the ELISA wasrepeated but with the addition of a defined amount of Fel d 1. SDS-PAGEexperiments as described above were conducted on all tested proteases toconfirm Fel d 1 degradation and ELISA results.

Results are shown in FIG. 2. As can be seen, complete suppression of Feld 1 binding to Fel d 1-specific antibodies was achieved with several ofthe subtilisin serine proteases, as well as trypsin andalpha-chymotrypsin; the thiol proteases papain, bromelain and ficin; theaspartic acid proteases mucorpepsin/rennin, pepsin, aspergillopepsin,endothiapepsin and chymosin; and the zinc-metalloproteases thermolysin(from Geobacillus) and endoproteinase Asp-N. Degradation of Fel d 1 tocompletion could be achieved by increasing the concentration of severalother of the proteases. SDS-PAGE further confirmed that these proteasesdegraded Fel d 1. Taken together, the ELISA and SDS-PAGE experimentsdemonstrate that antibody binding correlates with Fel d 1 degradation.For all tested proteases, the degree of Fel d 1 degradation, asestimated by SDS-PAGE, corresponded to the residual Fel d 1concentration, as determined by ELISA. While not intending to be limitedto a particular mechanism, these results suggest that the Fel d 1degradation products no longer contain epitopes that bind to the Fel d 1specific antibodies, and that protease-catalyzed degradation of Fel d 1results in the suppression of Fel d 1 binding to epitope-specific Fel d1 antibodies. Therefore, it is expected that Fel d 1 degradation byproteases can suppress IgE mediated allergic reactions. Most of thetested proteases were able to completely degrade Fel d 1 and suppressthe binding of the resultant degradation products to epitope specificantibodies. Representative proteases from these experiments capable ofcompletely degrading Fel d 1 (referred to below as “hit” proteases)include papain, subtilisin from B. licheniformis (bl subtilisin, PROTEX®6L), aspergillopepsin from A. oryzae (ao aspergillopepsin, PROTEX®50FP), endoproteinase Asp-N, bromelain, ficin, alpha-chymotrypsin,endothiapepsin from Cryphonectria parasitica (cp endothiapepsin,THERMOLASE®), pepsin and thermolysin from Geobacillus sp. (PROTEX® 14L).Table 3 summarizes the properties of these “hit” proteases, with detailsprovided in the following Examples.

TABLE 3 Summary of properties of “hit” proteases^(a) Keratinhydrolysis^(b) Cellulose hydrol^(d) Protease inactivation, TNBS Vitalityof keratinocytes^(c) Cellulose azure LD₅₀ ^(e) Name/tradename/typeKeratin azure Abs. 405 nm WST-1 Sulforhodamin B Abs. 575 nm IsopropanolTween 20 ® Group 1 hit proteases Papain (thiol) 9.0% ± 7.4%   0 ± 0.01103.4% ± 19.9% 99.1% ± 2.4%  0.00 ± 0.01 10.0%-12.5% 10%-20% Protex ® 6L(serine) 34.0% ± 33.5% 0.32 ± 0.02 110.5% ± 40.1% 107.5% ± 3.3%  0.00 ±0.01 2.5%-5.0% >30% Protex ® 50FP (aspartyl) 0.0% ± 0.1% 0.36 ± 0.06 92.8% ± 17.8% 108.2% ± 2.0%  0.08 ± 0.00  7.5%-10.0% >30%Endoproteinase Asp-N 0.43%^(f) 0.68 ± 0.03 24.7% ± 4.6% 3.7% ± 3.0%n.d.^(g)  7.5%-10.0% >30% (metallo) Group 2 hit proteases Bromelein(thiol) 43.2% ± 23.3% 0.28 ± 0.02 25.6% ± 5.2% 6.7% ± 1.4% 0.00 ±0.02 >25% 20%-30% Ficin (thiol) 33.1% ± 8.5%  0.21 ± 0.02 23.3% ± 4.0%6.7% ± 0.5% 0.00 ± 0.03 >25% 20%-30% Pepsin (aspartyl) 0.1% ± 0.6%   0 ±0.04  92.4% ± 19.6% 100.9% ± 3.5%  0.01 ± 0.01 5.0%-7.5% n.d.^(g)Protex ® 14L (metallo)  6.4% ± 13.6% 1.75 ± 0.04 23.5% ± 4.6% 5.1% ±0.7% 0.00 ± 0.02 5.0%-7.5% n.d.^(g) α-Chymotrypsin (serine)    22.3 ±17.8% 0.63 ± 0.01  95.8% ± 22.8% 101.3% ± 2.9%  0.00 ± 0.01 5.0%-7.5%n.d.^(g) Thermolase ® (aspartyl) 0.0% ± 0.2% 0.21 ± 0.04  89.1% ± 18.5%101.8% ± 5.2%  0.00 ± 0.01 2.5%-5.0% n.d.^(g) ^(a)For all tests,minimally required protease concentrations to degrade 125 μg ml⁻¹ of Feld 1 under optimal conditions were used (c.f. Table 2) and incubetiontook place overnight at 37° C. ^(b)Keratin hydrolysis by proteases wasdetermined either with keratin azure as surrogate substrate or directlyon keratin whereas hydrolysis products were determined by the TNBS assay(data extracted from FIG. 12). Activity on keratin azure was determinedas percentage of maximally hydrolysable substrate amount. Activity inthe TNBS assay was determined by the absorbance of the formed couplingproduct at 405 nm. See text for experimental details. ^(c)Vitality ofkeratinocytes in the presence of proteases was determined as describedin the text. Two different celitox staining methods were applied (WST-1and Sulforhodamin B) and vitality was determined as percentage ofvitality of keratinocytes that were not incubated with proteases.^(d)Cellulose hydrolysis by proteases was determined with celluloseazure as surrogate substrate. The assay was performed in duplicates andas proposed by the manufacturer (see text for details). The cellulosehydrolysis activity corresponds to the release of the azure dye, whichwas determined spectrophotometrically at 575 nm. As positive controlserved 2.5 units of cellulase from Aspergillus niger (Sigma-Aldrich.C1184), which resulted in a absorption of 0.28 ± 0.01. ^(e)Theinactivation of proteases by isopropanol and Tween 20 ® was determinedby estimation of the concentration range at which Fel d 1 degradation bythe proteases was reduced by more than 50% (LD₅₀). See text for detals.^(f)Determined only by a single experiment ^(g)n.d., not determined

Example 2

In all applications employing protease mediated degradation of Fel d 1,the cat and user would be exposed to protease activity. Filaments of theprotein keratin are abundant in keratinocytes in the cornified layer ofthe epidermis (skin), as well as in hair and nails of both humans andcats. Therefore, the identification of candidate Fel d 1 proteases withreduced proteolytic activity against keratin would be useful to addresssafety concerns in the development of products intended for human andanimal applications.

To characterize the substrate specificity of the active candidateproteases against physiologically relevant proteins, the ability ofcandidate Fel d 1 hit proteases to hydrolyze keratin was determined.Keratin azure (Sigma-Aldrich, K8500) is composed of azuredye-impregnated sheep's wool keratin and is used as a keratin substrateto quantify protease activity. Keratin azure was incubated overnightwith hit proteases under optimal conditions (as determined in theENZCHEK® protease assay) and at protease concentrations that were foundto completely degrade 125 μg ml⁻¹ Fel d 1 (Table 2). Degradation ofkeratin azure was then determined. In three repetitions, a significantdegradation activity was only reproducibly determinable with this assayfor the thiol proteases bromelain and ficin, which degraded 43.2%±23.3%and 33.1±8.5% of the applied keratin azure, respectively (Table 3). Theaspartyl proteases aspergillopepsin from A. oryzae (ao aspergillopepsin,PROTEX® 50FP), endothiapepsin from Cryphonectria parasitica (cpendothiapepsin, THERMOLASE®) and pepsin were not active, as theydegraded less than 1% of keratin azure in average (Table 3).

To further characterize the keratin degrading activity of hit proteases,an assay was performed that colorimetrically detects keratin degradationproducts by using a coupling reaction with trinitrobenzene sulfonic acid(TNBS) reagent. This assay enabled the determination of hydrolyzingactivity of all tested enzymes using keratin powder from sheep's wool assubstrate. Papain and pepsin showed no keratin degrading activity. Allother hit proteases were active against keratin substrate. The keratindegrading activity of Geobacillus thermolysin was at least two-foldhigher compared to all other tested proteases (FIG. 3). The results fromthe azure keratin assay described above correlated with the results fromthe TNBS-based keratin assay, with the exception of the two asparticacid proteases ao aspergillopepsin and cp endothiapepsin (Table 3). Forthis two aspartic acid proteases and Endoproteinase Asp-N, the bufferconditions may have affected the keratin degrading activity measurementsas determined by the TNBS assay. This is because the ammonium acetatebuffer (100 mM, pH 4.0) as well as the Endoproteinase Asp-N buffer(which was supplemented by the manufacturer) showed a basal level ofkeratin degrading activity in the assay in the absence of protease.Moreover, the TNBS-based assay determines proteolysis directly onkeratin whereas the keratin azure assay uses a derivatized keratinsubstrate, which might further explain inconsistencies between theresults of the two assays for some of the tested hit proteases.

As a model for living skin cells, the cytotoxic effects of the hitproteases were tested on keratinocytes. A primary cell line of humankeratinocytes was cultured to confluence in microplates and tested forcell viability after overnight incubation with the hit proteases atminimal concentrations found to completely degrade 125 μg ml⁻¹ Fel d 1(Table 2). Because cultured mammalian cells are highly susceptible tochanges in the composition and pH of cell culture medium, the proteaseswere directly diluted in the cell culture medium. After overnightincubation with the proteases, water-soluble tetrazolium WST-1 was addedto the cell culture (confluent layer and medium) (Cell proliferationreagent WST-1, Roche Diagnostics). The colorless WST-1 is reduced into ayellow dye (formazan) due to electron transfer catalyzed bymitochondrial dehydrogenases of vital cells. Thus, the formation ofyellow dye indicates cell vitality of both adherent and detached cells.Cell vitality of adherent cells alone was determined by staining withsulfrohodamin B after overnight incubation with proteases. The red colorformation is a measure of protein concentration, which correlates withthe adherent cell concentration. Cells that were not incubated withproteases served as a control. For all tested proteases, comparableresults were obtained for both assays (FIG. 4, Table 3).As can be seen,a relatively high cytotoxic effect (low remaining cell vitality) wasdetermined for bromelain, ficin, Geobacillus thermolysin andendoproteinase Asp-N. All other hit proteases (papain, aoaspergillopepsin, pepsin, cp endothiapepsin, bl subtilisin andalpha-chymotrypsin) had no significant effect on cell vitality ascompared to untreated cells (whose cell vitality corresponded to 100%).

For proteases that provoked cytotoxic effects on keratinocytes, skinirritating effects cannot be predicted with certainty because thekeratinocytes of the skin are protected by several layers of keratin.However, for proteases that did not provoke cytotoxic effects, skinirritating effects are unlikely.

The results of the cytotoxicity assay (FIG. 4) were less graduatedcompared to the results of the TNBS keratin degradation assay (FIG. 3).Proteases either had a significant cytotoxic effect or failed to elicitcytotoxicity. Nevertheless, correlations between the results of the twoassay were present. For example, papain and pepsin showed no effect ineither assay. On the other hand, the cytotoxic effects of bromelain,ficin, Geobacillus thermolysin and endoproteinase Asp-N on keratinocyteswere confirmed by the TNBS assay.

To further assess papain activity, keratinocytes were incubated in cellculture medium overnight with papain in concentrations 1000-fold greatercompared to the minimum required protease concentration shown in Table2. This resulted in significant cytotoxic effects, with 31% and 8% cellvitality as determined by the WST-1 and sulfurhodamin B assays,respectively. Most cosmetic applications do not require overnightincubation. Microscopy revealed that no significant cell detachmentoccurred after the first hour of incubation, even in the presence of1000-fold increased concentration of papain, indicating the absence ofcytotoxic effects at this earlier time point.

The effect of the protease preparations on cellulose fibers was testedwith cellulose azure (Sigma-Aldrich, St. Louis, Mo.), which is asubstrate analogous to keratin azure. This experiment was performed toestimate the effect of the hit protease preparations on cotton textiles.Proteolytic hydrolysis of cellulose was determined using cellulose azureas surrogate substrate. The assay was performed in duplicate and asproposed by the manufacturer. The cellulose hydrolysis activitycorresponds to the release of the azure dye, which was determinedspectrophotometrically at 575 nm. As a positive control, 2.5 units ofcellulase from Aspergillus niger (Sigma-Aldrich, C1184) was measured,resulting in an absorption of 0.28±0.01. Of the ten hit proteases, onlyao aspergillopepsin (PROTEX® 50FP) showed significant activity duringovernight incubation in the optimal Fel d 1 degradation buffer (Table2), corresponding to 28%±2% of the activity of the positive control(Table 3). The cellulase activity of this (or any) protease wasunexpected because proteases usually do not accept polysaccharides assubstrate. Presumably, this protease preparation is a crude extract ofprotease-overproducing Aspergillus oryzae and therefore contains otherenzymes that originate from the expression host, such as cellulases.

Most hit proteases showed no or only little keratinolytic activity orcytotoxic effects (FIGS. 3 and 4 and Table 3). Except for aoaspergillopepsin preparation (PROTEX® 50FP), no hit proteases wereactive on cellulose azure, indicating their inability to degrade cottontextiles. In addition, the use of high concentrations of proteases suchas papain in cosmetic products indicates that their use in applicationsto degrade Fel d 1 can be safe for both the user and the cat.

Example 3

Potential applications include cleansing agents or wipes, such as forthe cleaning of furniture or other surfaces contaminated with Fel d 1.Typical cleansing agents contain solvents, such as denatured ethanol orisopropanol, as disinfectants and to dissolve oil and grease. Theisopropanol concentration of a typical bath tub cleaner is 10 to 15%.However, solvents such as isopropanol can inactivate enzymes such asproteases. Therefore, hit proteases were tested for activity inisopropanol-containing media. Because cleaning agents are typicallydiluted by water, the Fel d 1 degrading activity of the hit proteaseswas tested in the presence of isopropanol at concentrations between 0and 12.5%.

Hit proteases were incubated overnight with 0, 2.5%, 5.0%, 7.5%, 10.0%and 12.5% of isopropanol at concentrations and under reaction conditionsoptimal for Fel d 1 degradation (Table 2). The samples were diluted andanalyzed by ELISA with an assay reading time point of 20 minutes. Asshown in FIG. 5, the residual Fel d 1 concentration represents thepercentage of Fel d 1 determined for the samples as compared to the Feld 1 for the respective buffer controls with maximal isopropanolconcentration but in the absence of protease. FIG. 5 shows averages andstandard deviations from three independent repetitions.

With the exception of bromelain and ficin, the activity of all proteaseswas reduced by at least 50% in the presence of 12.5% isopropanol. Thethiol proteases were the least sensitive to isopropanol. Even in thepresence of 12.5% of isopropanol, papain, bromelain and ficin degradedabout 40% to 70% of the added Fel d 1 (FIG. 5). In an additionalexperiment with 25% of isopropanol, bromelain and ficin degraded morethan 50% of Fel d 1. In contrast, the aspartic acid proteases were themost sensitive to isopropanol. Ao aspergillopepsin (PROTEX® 50FP),pepsin and cp endothiapepsin were inactivated by more than 90% in thepresence of 12.5% isopropanol. As a control, ELISA experiments revealedthat isopropanol alone was not found to significantly reduce Fel d 1concentration. Given this, bromelain or ficin appear to be particularlysuitable proteases to degrade Fel d 1 present in dust or on surfaces,using an isopropanol-containing cleaning agent. These proteases activelydegraded Fel d 1, even in isopropanol concentrations that exceed thosein typical cleaning applications.

Because contact with skin, hair and fur is minimal and transient, thehigher keratinolytic activities of bromelain and ficin, compared to theother tested proteases, are not expected to be detrimental. Moreover,bromelain and ficin are generally regarded as safe when used incosmetics (see above) or in nutrition processing (e.g., as meattenderizer).

Example 4

Potential applications for reducing Fel d 1 concentration on the fur ofthe cat include protease-containing shampoos or dry powders,foams/mousses among other compositions. A total of 67 mg of Fel d 1 percat has been estimated. Although only a small fraction of the allergenbecomes airborne, the large amount of Fel d 1 present on the catindicates the significant benefit afforded by reducing the amount of Feld 1 in or on the cat's fur and/or skin. The use of conventional petshampoos on cats has been reported to reduce Fel d 1 concentration inthe fur as well as the airborne Fel d 1 concentration in the cat's home.The efficacy of conventional shampoos can be increased by the additionof proteases as described herein.

Most shampoo formulations contain a mix of different surfactants, ofwhich anionic surfactants are the largest class. Non-ionic surfactantsare the second largest surfactant class, and have either polyether orpolyhydroxyl as a polar group to increase water solubility. Non-ionicsurfactants are widely used in topical applications because, they have areduced ability to cause irritation when compared to anionicsurfactants. This property makes non-ionic surfactants attractive forprotease-containing pet shampoos. TWEEN® 20 (polysorbate 20) isfrequently used as a non-ionic surfactant in nutrients and cosmetics. Itis registered as an approved food additive (E-432) in the European Unionand also in the 1NCI index (International Nomenclature of CosmeticIngredients). Because it is an approved food additive, TWEEN® 20 can beused as a shampoo ingredient, and would not pose safety issues if thecat accidentally ingests part of it during the washing procedure.

The effect of TWEEN® 20 on Fel d 1 degrading activity of the hitproteases was tested after overnight incubation with 0, 10%, 20%, and30% TWEEN® 20 and at protease concentrations and under reactionconditions optimal for Fel d 1 degradation as shown in Table 2. Thesamples were diluted and analyzed by ELISA with an assay reading timepoint of 20 minutes. The indicated residual Fel d 1 concentrationrepresents the percentage of Fel d 1 determined for the samples comparedto the Fel d 1 for the buffer controls with the respective TWEEN® 20concentrations but without protease. Averages and standard deviationswere determined from three independent repetitions. All tested hitproteases displayed tolerance to TWEEN® 20. Even at the highest testedconcentration of 30% TWEEN® 20 none of them was completely inactivated.At 10% TWEEN® 20, more than 50% of the 125 μg ml⁻¹ Fel d 1 was degradedfor all proteases (FIG. 6, Table 3). Given that a typical shampooformulation contains about 40% TWEEN® 20 and undergoes approximately20-fold dilution during application, the proteases would have to beactive in concentrations of approximately 2% TWEEN® 20 to retain Fel d 1degradation activity. Accordingly, all of the hit proteases be predictedas suitable for shampoo applications.

A pH neutral skin product has a pH of approximately 5.5, whichcorresponds to the pH on the skin. The Fel d 1 degrading activity of theaspartic acid protease ao aspergillopepsin (PROTEX® 50FP) was notaffected by TWEEN® 20 even at the highest tested concentration of 30%(FIG. 6). Although, this protease was found to have the highest activityat pH 4.0 (See Table 2), it still retained 46% activity at pH 6.0 in theENZCHEK® protease assay. In addition, ao aspergillopepsin (PROTEX®50FP), which did not show degradation activity when keratin azure wasused as substrate (Table 3), showed only minimal activity againstkeratin (FIG. 3) and had no significant cytotoxicity towardkeratinocytes (FIG. 4). These properties suggest that aoaspergillopepsin (PROTEX® 50FP) is suitable for use in aprotease-containing shampoo for Fel d 1 reduction.

Papain may also be a suitable alternative to ao aspergillopepsin(PROTEX® 50FP) in shampoo applications. Although papain is less tolerantto TWEEN® 20 than ao aspergillopepsin (PROTEX® 50FP), it remains activeat expected TWEEN® 20 concentrations of 2% during application (FIG. 6).Papain showed comparable Fel d 1 degradation activity at pH 7.4 and pH6.0, rendering it likely to be suitable for use in a pH neutral shampoo.Papain was not active against keratin in the TNBS assay (FIG. 3) and didnot induce significant cytotoxicity against keratinocytes (FIG. 4).Papain was not active on cellulose azure substrate, and is thereforelikely not to exhibit degradation activity against cotton textiles(Table 3). Papain is also used at high concentrations in nutrients (meattenderizer, BBQ sauces) and cosmetics (enzyme peeling) without requiringheat inactivation before use. Papain is likely applicable as a shampooingredient, even if the cat accidentally ingests the shampoo during thewashing procedure.

Example 5

Another application is a protease-containing product for use in a cat'smouth, including dentifrices, rinses, beverages, foods or treats, stripsor films. Such products can reduce Fel d 1 concentration in the mouth ofthe cat, which is a major source of the allergen. To test the activityof proteases under application-like conditions, a model for cat salivawas established. The preparation of artificial cat saliva was based onpublished artificial human saliva preparations (McKnight-Hanes &Whitford, 1992, Caries Res. 26: 345-350) (Table 4).

TABLE 4 Composition of artificial cat saliva Ingredients Contents perliter^(a) Methyl-p-hydroxybenzoate 2 g Sodium carboxymethyl cellulose 10g KCl 625 mg MgCl₂ × 6H₂O 59 mg CaCl₂ × 2H₂O 166 mg K₂HPO₄ 804 mg KH₂PO₄326 mg L-Cysteine 4.85 g Supplements for “extreme conditions” Fel d 1125 mg Skim milk 7.14 g Supplements for “normal conditions” Fel d 1 12.5mg Skim milk 4.50 g ^(a)The components are solved in water and pH isadjusted to 7.5 with KOH solution ^(b)Extreme and normal conditions arebenchmarks that refer to allergen and protein concentrations, which areabove expected and on average levels, respectively (see text fordetails)

The pH was adjusted to 7.5, which is the pH of cat saliva. Theconcentration of Fel d 1 in cat saliva was determined to be between 0.3to 45 μg/ml (whole cat population) and 2.2 to 12.4 μg/ml for anintermediate group, which is represented by one third of the wholepopulation. The maximum Fel d 1 concentration in the artificial catsaliva was defined as 125 μg ml⁻¹, as this concentration was used in allprevious experiments, although it exceeded the predicted maximum Fel d 1concentration in cat saliva by a factor of 2 to 3.

Skim milk, which contains approximately 35% protein, was used tosimulate the protein content in the saliva. Skim milk also containsother components such as carbohydrates, which are related to the complexcomposition of real saliva and are therefore predicted to render themodel more realistic. Human saliva from different individuals has beenreported to contain between 0.67 and 2.37 mg ml⁻¹ of protein (asdetermined by different techniques with bovine serum albumin asstandard). This estimate was used as an approximation for the proteincontent in cat saliva. A maximum protein content of 2.5 mg ml⁻¹ wasdefined for /the artificial cat saliva, which was represented by 7.14 mgml⁻¹ skim milk. As discussed above, cysteine has been found to activatecertain proteases for Fel d 1 degradation. The effect was most prominentfor thiol proteases, such as papain, bromelain and ficin (see above).

The effects of different concentrations of Fel d 1 and skim milk, aswell as the requirement of cysteine for Fel d 1 degradation inartificial saliva, were tested using the hit protease papain withovernight incubation at 37° C. (FIG. 7). Papain was used at the minimumconcentration required to completely degrade 125 μg ml⁻¹ Fel d 1 underoptimal conditions, i.e. in sodium phosphate buffer at pH 7.8 (Table 2).Similar to the previous experiments conducted in buffer, 40 mM ofL-cysteine was shown to be essential for efficient degradation of Fel d1 by papain in artificial cat saliva (FIG. 6); however, this requirementcould be satisfied by other components of the formulation, especially inan edible compositions such as a food, beverage or treat.

Although, reaction conditions in artificial cat saliva differedsignificantly from the optimal reaction conditions reported in Table 2,most of the added 125 μl g ml⁻¹ Fel d 1 was degraded during overnightincubation (in the presence of cysteine) for skim milk concentrations upto 714 μg ml⁻¹ (FIG. 7). Nevertheless, Fel d 1 degradation activitydecreased with increasing skim milk concentration, probably due to theincreased protein concentration. Skim milk proteins predominantlyconsist of casein, and casein was shown to be hydrolyzed by papain viathe ENZCHEK® protease assay, which uses labeled casein as substrate(Table 2). Casein and Fel d 1 thus represent competing substrates forthe degradation by papain. Therefore, the concentration of papain wouldneed to be increased to completely degrade Fel d 1 at the highest skimmilk concentration (and in the presence of cysteine). Moreover, anincreased papain concentration would likely result in a faster Fel d 1degradation, which would be preferred in order to degrade Fel d 1 in thesaliva within the time frame in which the cat consumes theprotease-containing pet food or beverage, or is otherwise orally exposedto a protease-containing formulation.

Two types of artificial cat saliva were developed for further testing(Table 4). The first type represented “extreme conditions,” andcontained 125 μg ml⁻¹ of Fel d 1 (2 to 3-fold higher concentration thanthe maximum concentration determined in cat saliva) and 7140 μg ml⁻¹ ofskim milk (corresponding to the maximally determined protein content inhuman saliva). These allergen and protein concentrations are aboveexpected levels and are provided to estimate the surplus activity ofproteases in a pet food application. The second type represented “normalconditions,” and contained 12.5 μg m⁻¹ of Fel d 1 (representing theupper border of Fel d 1 concentrations as determined in the intermediatecat group) and 4.5 μg ml⁻¹ of skim milk (corresponding to the averageprotein concentration in human saliva of 1.6 μg mr⁻¹). These allergenand protein concentrations represent an average level expected in thesaliva of most cats.

To identify the protease activity required to degrade Fel d 1 inartificial cat saliva (under normal and extreme conditions) within arelatively short period of time, the hit proteases papain, bl subtilisin(PROTEX® 6L), ao aspergillopepsin (PROTEX® 50FP) and endoproteinaseAsp-N were evaluated by additional kinetic analysis at higher enzymeactivities. Preliminary experiments revealed that up to a 1000-foldincrease of papain, from 4.5 μg ml⁻¹ (corresponding to the minimumconcentration required to completely degrade Fel d 1 under optimalconditions during an overnight incubation, Table 2) to 4500 μg ml⁻¹, isrequired to completely degrade Fel d 1 in artificial cat saliva underextreme conditions within 1 hour of incubation.

Therefore, for bl subtilisin, amounts that were 10-fold, 100-fold, and1000-fold greater than 9.5 μg bl subtilisin, which was the minimumconcentration of protease required to degrade 125 μg ml⁻¹ Fel d 1 underoptimal conditions during an overnight incubation (Table 2) were testedin artificial cat saliva under extreme conditions for Fel d 1degradation after one and 20 hours of incubation at 37° C. (FIG. 8).Thus, 125 μg ml⁻¹ natural Fel d 1 was incubated for 0 min, 1 hour or 20hours (overnight) with 0, 9.5, 95, 950 and 9500 μg ml⁻¹ of bl subtilisinat 37° C. in artificial cat saliva (extreme conditions, c.f. Table 4).The reaction was stopped by the addition of 1 mM PMSF. Samples werediluted and analyzed by ELISA with an assay reading time point of 30minutes. Taking into account the dilution, a maximum concentration of 12ng ml⁻¹ of Fel d 1 was expected (i.e. without accounting for thepossibility of degradation). Averages and standard deviations weredetermined from two independent repetitions of the experiment. Similarto the preliminary experiments with papain, a 1000-fold higher blsubtilisin concentration was required to completely degrade Fel d 1within 1 hour in artificial cat saliva under extreme conditions (FIG.8).

To further determine the efficacy of the proteases in 1000-fold excess,Fel d 1 degradation by papain and bl subtilisin was kinetically analyzedin artificial cat saliva under normal and extreme conditions at shorterincubation times (FIG. 9). Papain and 9.5 mg ml⁻¹ of bl subtilisin wereincubated for 0, 5, 10, 15 and 60 min with Fel d 1 in artificial catsaliva at 37° C. under normal and extreme conditions (Table 4).Reactions were stopped by addition of 100 μM E64 (for papain) and 1 mMPMSF (for bl subtilisin). Samples were diluted and analyzed by ELISAwith an assay reading time point of 30 minutes. The residual Fel d 1concentration represents the percentage of Fel d 1 determined for thesamples as compared to the Fel d 1 for the respective buffer controlswithout protease. Averages and standard deviations were determined fromthree independent repetitions. Most of the Fel d 1 was degraded alreadyafter 5 min with bl subtilisin (9.5 mg ml⁻¹) in artificial cat salivaunder both normal and extreme conditions. The experiments revealed thatbl subtilisin was somewhat more effective than papain. However, undernormal conditions, which correspond to the average composition of catsaliva, more than 80% of Fel d 1 was degraded by papain (4.5 mg ml⁻¹)within 5 minutes (FIG. 9). The apparent Fel d 1 degradation activityunder these conditions at 0 min could potentially be explained byresidual activity despite immediate addition of E64 inhibitor toterminate the reaction. This effect was avoided in the followingexperiments by the addition of both inhibitors, E64 and PMSF, toterminate activity of papain and bl subtilisin combinations (FIGS.10-12).

In the context of a pet food application, where a cat with average Fel d1 and protein concentration in the saliva takes about five to tenminutes to eat food, both papain and bl subtilisin, present in theconcentrations tested, would be predicted to degrade the allergen bymore than 80% within 5 minutes. Both concentrations tested, 4.5 mg ml⁻¹papain and 9.5 mg ml⁻¹ bl subtilisin, respectively, are present atconcentrations that are in massive excess over the applied Fel d 1 (125μg ml⁻¹). However, this excess enzyme promotes the accelerateddegradation of Fel d 1, such that most of it is degraded within a fewminutes.

Different combinations of papain and bl subtilisin were tested toidentify synergistic effects on Fel d 1 degradation, which would permita decrease in the total concentration of proteases, while maintainingefficacy of allergen degradation comparable to that of a single proteasein the final cat food application. The thiol protease papain and the blsubtilisin belong to different mechanistic families of proteases andtherefore have different substrate specificities. While not intending tobe limited to a particular mechanism, it is thought that the twoproteases could act synergistically by mutually uncovering new cleavagesites on the Fel d 1 polypeptide chain, resulting in a faster and moreeffective degradation compared to the degradation achieved by using onlya single protease.

The two proteases were tested in combination for their ability tosynergistically degrade Fel d 1 in artificial cat saliva at theconcentrations previously tested for each protease alone (4500 μg ml⁻¹of papain and 9500 μg ml⁻¹ of bl subtilisin, FIG. 9). In addition, Fel d1 degradation was tested at protease concentrations reduced by a factorof 10, 100 and 1000. 450 μg ml⁻¹ papain and 950 μg ml⁻¹ bl subtilisinwere minimally required to completely degrade Fel d 1 within one hour(FIG. 10). At the combination with highest tested proteaseconcentrations (4500 μg ml⁻' papain and 9500 μg ml⁻¹ bl subtilisin)complete Fel d 1 degradation was achieved after 10 minutes (FIG. 10).This effect was comparable to the effect of 9500 μg ml⁻¹ bl subtilisinalone (FIG. 9).

To evaluate the contribution of bl subtilisin, its concentration wasvaried from 1188 to 9500 μg ml⁻¹ while keeping the concentration ofpapain constant (at 4500 μgml⁻¹) (FIG. 11). Only a small difference inFel d 1 degradation was observed when 4500 μg ml⁻¹ papain was combinedwith 9500 or 4750 μg ml⁻¹ bl subtilisin. Therefore, a combination of4500 μg ml⁻¹ papain and 4750 μg ml⁻¹bl subtilisin would be sufficient todegrade most Fel d 1 present in cat saliva within 10 minutes, even underextreme conditions. Under normal conditions, which is the case for mostcats, the combination with the lowest bl subtilisin concentration (1188μg ml⁻¹) is sufficient to degrade about 90% of Fel d 1 within 10minutes.

To evaluate the contribution of papain, its concentration was variedfrom 565 to 4500 μg ml⁻¹ while keeping the concentration of blsubtilisin constant at the lowest previously tested concentration (1188μg ml⁻¹) (FIG. 12). Under extreme conditions, a reduction of 80% of Feld 1 in 10 minutes was only achieved when combined with 4500 μg ml⁻¹papain (FIG. 12). In contrast, under normal conditions, the testedpapain concentration did not significantly influence Fel d 1 degradationafter 10 minutes of incubation. Therefore, the minimum proteasecombination of 565 μg ml⁻¹ papain and 1188 μg ml⁻¹ bl subtilisin wouldbe sufficient to reduce Fel d 1 content in the saliva of an average catby approximately 80% within 10 minutes of incubation, e.g. while the catis eating the pet food or treat, or drinking a beverage containing theproteases, or otherwise being orally exposed to the protease-containingformulation.

Example 6

A wipe prototype containing functional proteases was developed andtested for inactivation of Fel d 1. A batch of cotton wipes (20 cm×20cm) containing active PROTEX® 6L was manufactured and tested for Fel d 1inactivation. The wipe was successful in degradation of Fel d 1. Thespecifics are discussed in detail below.

First, the concentration of PROTEX® 6L required for the wipe wasdetermined. The required enzyme concentration is dependent on the amountof Fel d 1 that has to be inactivated by the prototype. Based on thedata provided by Nestl—Purina, the amount of Fel d 1 in the environmentthat should be digested by the wipe prototype is approx. 25 ng/cm2(=0.25 mg/m2).

For a wipe prototype, it is not possible to simply set the desiredenzyme concentration since the binding capacity for the enzyme might belimited by the available binding/cross-linking sites of the solidmaterial. Therefore the aim was to maximize the amount of immobilizedPROTEX® 6L.

Table 5 summarizes the application- and safety-relevant properties ofthe hit proteases analyzed herein based on the data from Table 3. It wasdiscovered that protease PROTEX® 6L combines all characteristicsrequired for development of application prototypes, including those ofExamples 6-10: It shows only weak effects on keratin hydrolysis, noeffects on viability of keratinocytes, no cellulose activity but hightolerability towards non-ionic detergents. Therefore, PROTEX® 6L wasused for the development of the present prototypes (wipes, cat litter,shampoo, cleansing liquid).

TABLE 5 Summary of properties of hit proteases Cytotoxicity DetergentKeratin In Toler- Cellulose Enzyme Hydrolysis keratinocytes abilityhydrolysis Papain No No Low No Protex ® 6L Low No High No Protex ® 50FPLow No High Medium Endoproteinase Asp-N Medium Strong High No BromelainLow Strong Medium No Ficin Low Strong Medium No Pepsin No No n.d. NoProtex ® 14L Strong Strong n.d. No Alpha-chymotrypsin Medium No n.d. NoThermolase ® Low No n.d. No n.d.: not determined

Immobilization of PROTEX® 6L on Cotton Wipes

Fabrication of Cotton Wipes

From the numerous types of cotton fabrics, molton was chosen for thefabrication of the wipes. Molton fabric consists of 100% cotton fibers.Its roughened surface and its high water absorption make it to aversatile cleaning tissue. The size of one final wipe is approx. 20cm×20 cm.

Cross-Linking Systems for Immobilizing PROTEX® 6L

In theory, immobilization of enzymes on solid material can be achievedby covalent coupling of the enzyme via a chemical reaction. However,there are no well-established protocols available that describe thecoupling of PROTEX® 6L to cotton fibers.

A first screening of conditions for the immobilization of PROTEX® 6Lused not only the enzyme and carrier (cotton wipe) but also twocross-linkers since the carrier material (cotton) does not possessgroups that readily form covalent bonds. The cross-linker used is a2-component system: a diamine (pentaethylenehexamine, PEHA) and adialdehyde (glutaraldehyde, Ga.). They are combined in a fixed ratio andreference is mostly made to the glutaraldehyde concentration to indicatethe amount used in proportion to the enzyme.

The 2-component cross-linking system has to match the amount of protein.It was found that the amount used in proportion to PROTEX® 6L iscritical as too low amounts will not cross-link all the enzyme or willresults in unstable formulations. Too high amounts will giveovercross-linking reducing conformational freedom and subsequentlyreducing the activity of the enzyme.

Enzymatic Assay for Determining Immobilization Efficiency

In order to monitor the success of the different cross-linkingconditions and protocols, the enzymatic activity after cross-linking andwashing was determined. At that stage, it is inconvenient and timeconsuming to directly determine the Fel d 1 degrading activity ofcross-linked PROTEX® 6L by ELISA. To allow a much faster screening ofcross-linking conditions, two other enzymatic assays were chosen: TheNPA assay, which is based on hydrolysis of the substratep-nitrophenylacetate by the protease or the ELU test, which is based onthe hydrolysis of ethyl lactate. After establishing the final protocolfor cross-linking PROTEX® 6L on small cotton wipes, large scaleimmobilizations on entire wipes will be performed. Then the capabilityof degrading Fel d 1 and the enzymatic stability will be directly testedon the target substrate Fel d 1 by ELISA.

Final Production of Immobilized PROTEX® 6L on Cotton Wipes

Cross-linker amounts and conditions as well as enzyme loadings amountsand conditions were experimentally determined. After which, big-scalereactions were performed to cross-link PROTEX® 6L on complete wipes (20cm×20 cm). In detail, liquid PROTEX® 6L (9.24 ml of 4600 ELU/ml PROTEX®6L) was mixed with 83.2 ml phosphate buffer (100 mM, pH 6.5) andglutaraldehyde was added (133 mM final concentration). Cross-linking ofthe enzyme occurred by incubating the wipe in the reaction mixtureover-night at room-temperature. The wipe was washed with water and theliquid is squeezed out and washed again with water and 3% PEG. Finally,the wipe is dried with a stream of air (21° C.) for 5 h.

In summary, PROTEX® 6L was immobilized on 10 cotton wipes, another 10control wipes were produced the same way, with the exception that noenzyme was added to the reaction mixture. The wipes were then tested forapplication. The capability of Fel d 1 inactivation was determined andshown in Example 10 (Table 6).

Example 7

Cat litter prototypes containing functional proteases were developed andtested for inactivation of Fel d 1. Specifically, 260 g cat litter(perlite) containing active PROTEX® 6L was manufactured and tested forFel d 1 inactivation. The cat litter was successful in degradation ofFel d 1. The specifics are discussed in detail below.

First, the concentration of PROTEX® 6L required for the prototype wasdetermined. The required enzyme concentration is dependent on the amountof Fel d 1 that has to be inactivated by the prototype. Based on thedata provided by Nestl—Purina, the amount of Fel d 1 that should beinactivated by the cat litter is approx. 250 mg.

In case of cat litter, it is not possible to simply set the desiredenzyme concentration since the binding capacity for the enzyme might belimited by the available binding/cross-linking sites of the solidmaterial. Therefore the aim was to maximize the amount of immobilizedPROTEX® 6L.

Immobilization of PROTEX® 6L on Cat Litter

Immobilization on Bentonite

“Purina Tidy Cats non clumping cat litter Instant Action” was tested forthe immobilization of PROTEX® 6L. The cat litter is based on the claymineral bentonite. PROTEX® 6L was diluted with water and added tobentonite to check possible immobilization conditions and absorption ofthe enzyme. However, adding water to PROTEX® 6L dilutes down the 50%glycerol present in this enzyme mixture and makes the bentonite fallapart.

Another immobilization test on bentonite was performed with minimal useof liquid. To this purpose small amounts of PROTEX® 6L that could bereadily absorbed were added directly to the bentonite. Besides theenzyme and carrier two cross-linkers were added since the carriermaterials do not possess groups that readily form covalent bonds. Thecross-linker used is a 2-component system: a diamine(pentaethylenehexamine, PEHA) and a dialdehyde (glutaraldehyde, Ga.),which are combined in a fixed ratio. It turned out that a maximumloading is 150-200 μl of liquid per gram of bentonite. The liquid has tobe administered individually to each particle. Due to the fact that theparticles fall apart, no work-up is possible after immobilization. Thisis not an immediate drawback but dust with enzyme will get airborneafter the bentonite has been wet and dried up again. However, PROTEX® 6Lon this carrier lost all activity after a couple of days, even when dry.In conclusion, the tested cat litter was not suited for theimmobilization of PROTEX® 6L.

Immobilization on Sand

Sand with a particle size of 1.2 mm was tested as another carrier whichcould be principally be mixed with bentonite. Again two cross-linkerswere used besides the enzyme. Sand was used to demonstrate the viabilityof the approach where the enzyme is immobilized on a second carrier(sand), which could then be mixed with other cat litter particles. Amaximum loading of 200 μl PROTEX® 6L was used per gram of sand. However,the activity and recovery was relatively low, most likely because sandlacks pores and has a small surface area. Leaching of the enzyme is lowand thus sand is in principle a suitable material for covalent linkage.

Immobilization on Perlite

Perlite was chosen as a third carrier. Due to its particle size of 2.8-6mm, it could also principally be mixed with bentonite. Again twocross-linkers (GA and PEHA) were used besides the enzyme to inducecovalent bonding. First immobilization experiments showed that therecovery as well as the leaching of the enzyme PROTEX® 6L is relativelygood for enzyme loadings in the lower range. Enzyme loadings above 500μl /g give significant coloration of the perlite particles but withloadings in the lower range it maintains a bright white color.

Final production of immobilized PROTEX® 6L on perlite

Clearly, for cross-linking of PROTEX® 6L to cat litter material, perliteturned out to be superior to sand and bentonite. Perlite permits highenzyme loading, while keeping enzyme leaching low. Moreover, itsparticle size allows homogenous mixing with other cat litter material.Therefore, perlite was found to be an excellent material forcross-linking of PROTEX® 6L and finally 260 g perlite containing PROTEX®6L were produced.

In detail, liquid PROTEX® 6L (60 ml of 4600 ELU/ml PROTEX® 6L) was mixedwith 472.8 ml cold water, PEHA (adjusted to pH 7.0, 20 mM finalconcentration, 4° C.) and glutaraldehyde (133 mM final concentration)wasadded, mixed well and added to 300 g perlite. Cross-linking of theenzyme occurred by incubating perlite particles in the reaction mixtureovernight at room-temperature. Perlite was washed with water, 5 mMphosphate buffer, and water with 3% PEG. Finally, perlite particles weredried over-night at room temperature.

In summary, PROTEX® 6L was immobilized on 260 g perlite, another smallcontrol sample (1 g perlite) was produced the same way, with theexception that no enzyme was added to the reaction mixture. Perlite wasthen tested for application. The capability of Fel d 1 inactivation wasdetermined and shown in Example 10 (Table 6).

Example 8

Cleansing liquid prototypes containing functional proteases weredeveloped and tested for inactivation of Fel d 1. Specifically, 1 Literof cleansing liquid and 50 ml of enzyme concentrate containing activePROTEX® 6L were manufactured and tested for Fel d 1 inactivation. Theprototypes were successful in degradation of Fel d 1. The specifics arediscussed in detail below.

First, the concentration of PROTEX® 6L required for every prototype wasdetermined. The required enzyme concentration is dependent on the amountof Fel d 1 that has to be inactivated by the respective prototype. Basedon the data provided by Nestl—Purina, the amount of Fel d 1 in theenvironment that should be digested by the prototypes is approx. 25ng/cm2 (=0.25 mg/m2).

For the determination of PROTEX® 6L concentration that is required fortotal Fel d 1 digestion, the activity of a PROTEX® 6L dilution serieswas analyzed by ELISA. 3.8 U/ml PROTEX® 6L (Units are based onethyl-L-lactate assay (ELU), determined by manufacturer Genencore) arecapable to completely digest 2.5 μg Fel d 1 under optimal reactionconditions (Tris-HCl buffer, pH 7.8). Based on these calculations,including the initial calculations for the shampoo in Example 9, theconcentration of PROTEX® 6L was adjusted accordingly for the cleansingliquids.

Development of Surface Cleanser Containing PROTEX® 6L

Based on the Fel d 1 concentration on hard surfaces, the required amountof PROTEX® 6L was determined and incorporated into the basic cleansingliquid formulation (KAR-001). In detail, water (960 g) was added to abeaker glass, then 10 g phenoxyethanol (preservative), followed by 30 gZusolat 1008/85 (fatty alcohol available from Zschimmer & Schwarz GmbH &Co KG) and 10 g EUXYL® PE 9010 were added and stirred. If necessary pHhad to be adjusted to 8.0 by using NaOH. To this basic formulation(KAR-001) PROTEX® 6L (9 ml of 4600 ELU/ml PROTEX® 6L) was added(KAR-001+E). Finally, 1 Liter of surface cleanser KAR-001 containingPROTEX® 6L (KAR-001+E) as well as 2×1 Liter of surface cleanser withoutPROTEX® 6L (KAR-001-E) were produced and tested for application. Thecapability of Fel d 1 inactivation was determined and is shown inExample 10 (Table 6).

Development of an Enzyme Concentrate Containing PROTEX® 6L

In addition to the ready-to-use surface cleanser, which already containsPROTEX® 6L, one could envision another application, which is based onmixing enzyme and liquid surface cleanser just before use.

The advantage is that the enzyme could be mixed with any other surfacecleanser, which could either be provided by the customer. In additionstoring the enzyme in an optimized liquid formulation and not in surfacecleanser formulation could stabilize the enzyme and lead to increasedenzymatic stability. Therefore, an enzyme concentrate consisting of 20 xhigher concentrated PROTEX® 6L (in comparison to the ready-to-usesurface cleanser KAR-001+E) and enzyme stabilizer propylene glycol wasdeveloped. The capability of Fel d 1 inactivation (after dilution of the20×KAK-001+E in 1×surface cleanser) was determined and is shown inExample 10 (Table 6).

Example 9

Shampoo prototypes containing functional proteases were developed andtested for inactivation of Fel d 1. Specifically, two cat shampoos(leave-on formulation, 1 Liter each), containing active PROTEX® 6L wasmanufactured and tested for Fel d 1 inactivation. All prototypes weresuccessful in degradation of Fel d 1. The specifics for each prototypeare discussed in detail below.

First, the concentration of PROTEX® 6L required for the prototype wasdetermined. The required enzyme concentration is dependent on the amountof Fel d 1 that has to be inactivated by the prototype. The Fel d 1amount on the cat is approx. 2 μg/mg cat hair (data provided byNestl—Purina). Since the weight of hair per cat is in average 121 g(Avner DB et al., J Allergy Clin Immunol, 1997) the total amount of Feld 1 on the cat that should be inactivated by the shampoo is approx. 250mg

For the determination of PROTEX® 6L concentration that is required fortotal Fel d 1 digestion, the activity of a PROTEX® 6L dilution serieswas analyzed by ELISA. 3.8 U/ml PROTEX® 6L (Units are based onethyl-L-lactate assay (ELU), determined by manufacturer Genencore) arecapable to completely digest 2.5 μg Fel d 1 under optimal reactionconditions (Tris-HCl buffer, pH 7.8). Next, the activity of PROTEX® 6Lin cat shampoo KFS-002 (see below) was analyzed to determine theactivity of PROTEX® 6L in a shampoo formulation. KFS-002 was chosen asmodel for initial calculations of PROTEX® 6L activity in a liquidformulation. It was determined by ELISA that in KFS-002 38.4 U/mlPROTEX® 6L is required for total inactivation of Fel d 1 (which isapprox. 10×higher than in the optimal reaction buffer).

Development of Shampoos Containing PROTEX® 6L

Several foaming formulations were tested based on amphotericcocamidopropylbetain (betaine), anionic sodium laurylethersulfate (SLE),anionic sodium lauroyl sarcosinate (SLS), anionic disodium/sodium cocoylglutamate and non-ionic decyl glucoside but not every formulation didprovide acceptable results.

Based on the Fel d 1 concentration on the cat, the required amount ofPROTEX® 6L was determined and incorporated into two basic shampooformulations (KFS-002 and KFS-004a).

In detail, water (980 g) was added to a beaker glass, then 10 gphenoxyethanol (preservative), followed by 10 g betaine (KFS-002) or 10g SLS (KFS-004a) and 10 g PERLASTAN® L30 were added and stirred. Ifnecessary, pH had to be adjusted to 5.5 (KFS-002) or 7.5 (KFS-004a) byusing NaOH. To this basic formulation (KFS-002/004a) 9 ml PROTEX® 6L (of4600 ELU/ml PROTEX® 6L) was added (KFS-002/004a+E). Finally, 1 Liter ofshampoo KFS-002 and 1 Liter of shampoo KFS-004a containing PROTEX® 6L(KFS-002/004a+E) as well as 2×1 Liter of shampoos without PROTEX® 6L(KFS-002/004a-E) were produced and tested for application. Thecapability of Fel d 1 inactivation was determined and is shown inExample 10 (Table 6).

Example 10

The prototypes of Examples 6-9 were studied for inactivation of Fel d 1.The data is presented in table 6. Clearly, all developed prototypes arecapable to inactivate application-relevant Fel d 1 amounts.

TABLE 6 Prototype Capability of Fel d 1 inactivation KAK-001 ~57-71 mgFel d 1/10 ml Enzyme Conc. KAR-001 ~150-203 mg Fel d 1/10 ml SurfaceCleanser KFS-002 ~105-156 mg Fel d 1/10 ml Shampoo KFS-004a ~18-23 mgFel d 1/10 ml Shampoo Wipe ~1.09 mg Fel d 1/wipe/usage Cat Litter ~9.3mg Fel d 1/100 mg

In the specification, there have been disclosed typical preferredembodiments of the invention and, although specific terms are employed,they are used in a generic and descriptive sense only and not forpurposes of limitation, the scope of the invention being set forth inthe claims. Numerous modifications and variations of the invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

We claim:
 1. A formulation for reducing or eliminating allergenic Fel d1 from an environment, comprising at least one protease that interactswith the Fel d 1 and substantially degrades allergenic epitopes on theFel d
 1. 2. The formulation of claim 1, wherein at least one protease isselected from serine proteases, thiol proteases, aspartyl proteases andzinc metalloproteases.
 3. The formulation of claim 2, wherein the serineprotease is selected from subtilisin, proteinase K, trypsin,alpha-chymotrypsin, endoproteinase Glu-C and endoproteinase Lys-C. 4.The formulation of claim 3, wherein the subtilisin is from a species ofBacillus selected from B. licheniformis, B. clausii, B. haloudurans, B.lentus, B. amyloliquefaciens or B. subtilis.
 5. The formulation of claim2, wherein the thiol protease is selected from bromelain, papain andficin.
 6. The formulation of claim 2, wherein the aspartyl protease isselected from chymosin, endothiapepsin, mucorpepsin/rennin, pepsin andaspergillopepsin.
 7. The formulation of claim 2, wherein the zincmetalloprotease is selected from thermolysin and endoproteinase Asp-N.8. The formulation of claim 7, wherein the thermolysin is from Bacillusthermoproteolyticus rokko or Geobacillus sp. and the endoproteinaseAsp-N is from Flavobacterium menigosepticum.
 9. The formulation of claim1 wherein the protease reduces Fel d 1 binding to anti-Fel d 1antibodies by at least 90% under assay conditions pertinent to theprotease as set forth in Table 2, as measured by ELISA.
 10. Theformulation of claim 9, wherein the protease is selected from one ormore of subtilisin, trypsin, alpha-chymotrypsin , bromelain, papain,ficin, chymosin, endothiapepsin, mucorpepsin/rennin, pepsin,aspergillopepsin, thermolysin and endoproteinase Asp-N.
 11. Theformulation of claim 1, wherein the proteases are selected from one ormore of papain, subtilisin from B. licheniformis, aspergillopepsin fromA. oryzae, endoproteinase Asp-N, bromelain, ficin, alpha-chymotrypsin,endothiapepsin from Cryphonectria parasitica, pepsin and thermolysinfrom Geobacillus sp.
 12. The formulation of claim 11, wherein theproteases are selected from one or more of papain, subtilisin from B.licheniformis, aspergillopepsin from A. oryzae and endoproteinase Asp-N.13. The formulation of claim 1, wherein the protease reduces Fel d 1binding to anti-Fel d 1 antibodies by at least 50% under assayconditions pertinent to the protease as set forth in Table 2 andcontaining 7.5% isopropanol, as measured by ELISA or wherein theprotease reduces Fel d 1 binding to anti-Fel d 1 antibodies by at least50% under assay conditions pertinent to the protease as set forth inTable 2 and containing 10% poysorbate 20, as measured by ELISA.
 14. Theformulation of claim 13, wherein the protease is selected from one ormore of aspergillopepsin from Aspergillus oryzae, endoproteinase Asp-N,subtilisin from B. licheniformis, papain, bromelain and ficin.
 15. Theformulation of claim 1, further comprising an additive that enhances theefficiency of the protease in degrading the Fel d 1, wherein theadditive is cysteine or calcium salt/ions (Ca²⁺), or a combination ofcysteine and calcium salt/ions (Ca²⁺).
 16. The formulation of claim 1,disposed within a composition selected from: liquid, solid or powdercleaning agent, spray, moist cloth, wipe, sponge, water-dissolvabletablet, filter, food, oil or water supplement, vacuum cleaner filter oradditive, granule, detergent, carpet and room deodorizer, litter, litteradditive, mitt, additive for non-woven products, washing machine pod(tablet) and multi-chamber liquid tablet.
 17. The formulation of claim1, wherein the protease is recognized as safe for use in foods andcosmetics.
 18. The formulation of claim 17, disposed within acomposition selected from soap, shampoo, rinse, powder, spray, gel,mousse, foam, conditioner, lotion, collar, dispersant or moist mitt orwipe suitable for applying to the skin, hair or fur.
 19. The formulationof claim 17, disposed within an edible composition.
 20. The formulationof claim 17, wherein the protease has substantially no interaction withsubstances selected from: keratin, collagen, elastin, fibronectin, otherproteins and fibers or fabric.
 21. A method of making a formulation forreducing or eliminating allergenicity of Fel d 1, comprising combiningat least one protease that interacts with the Fel d 1 and substantiallydegrades allergenic epitopes on the Fel d 1 with a medium in which theprotease is active or can be made active prior to use.
 22. A method ofreducing or eliminating allergenic Fel d 1 from the environment,comprising contacting an element of the environment where Fel d 1 ispresent with a formulation comprising at least one protease thatinteracts with the Fel d 1 and substantially degrades allergenicepitopes on the Fel d 1, thereby reducing or eliminating allergenic Feldl from the environment.
 23. The method of claim 22, wherein the Fel d 1is present on an inanimate surface and the formulation is applied to thesurface.
 24. The method of claim 22, wherein the Fel d 1 is airborne andthe formulation is contacted with the air.
 25. The method of claim 22,wherein the Fel d 1 is present in or on an animal that produces Fel d 1and the formulation is applied to the portion of the animal on which theFel d 1 is present.
 26. The method of claim 25, wherein the Fel d 1 ispresent on the hair, fur or external skin of the animal or is present inthe mouth of the animal.
 27. An article of manufacture comprising aformulation that includes at least one protease that interacts with theFel d 1 and substantially degrades allergenic epitopes on the Fel d 1,and instructions for its use in reducing or eliminating allergenic Feldl from the environment.
 28. The article of manufacture of claim 27,formulated for application to an inanimate surface.
 29. The article ofmanufacture of claim 28, selected from liquid, solid or powder cleaningagent, spray, moist cloth, wipe, sponge, water-dissolvable tablet,detergent, carpet or fabric deodorizer, litter, litter additive, mitt,additive for non-woven products, washing machine tablet.
 30. The articleof manufacture of claim 28, comprising a filter formulated forcontacting airborne Fel d
 1. 31. The article of manufacture of claim 27,formulated as a shampoo, conditioner, rinse, gel, mousse, spray, lotionor powder for application to hair, fur or external skin of the animal.32. The article of manufacture of claim 27, formulated as a dentifrice,film, food, treat, or additive to the animal's food or water forapplication to the mouth of the animal.